Analyzing the radiation of the proenkephalin gene in tetrapods: cloning of a Bombina orientalis proenkephalin cDNA

Analyzing the Radiation of the Proenkephalin Gene in Tetrapods: Cloning of a Bombina orientalis Proenkephalin cDNA: A proenkephalin cDNA was cloned from the brain of the anuran amphibian, Bombina orientalis (Family: Discoglossidae). This cDNA is 1358 nucleotides in length, and contains an open reading frame that codes for 251 amino acids. Within the open reading frame there are seven opioid (YGGF) sequences. There were five Met-enkephalin (YGGFM) sequences that are flanked by sets of paired basic amino acid proteolytic cleavage sites and two C-terminally extended Met-enkephalin sequences: YGGFMRGY and YGGFMRF. No Leu-enkephalin sequences were found in B. orientalis proenkephalin. It was possible to align the amino acid sequences of proenkephalin from several vertebrate taxa (human, Australian lungfish, B. orientalis, Xenopus laevis, Spea multiplicatus) by inserting a minimum of nine gaps. This alignment was then used to analyze the corresponding nucleotides for each proenkephalin sequence using maximum likelihood. This analysis yielded a single tree. In this tree, the Australian lungfish sequence was the outgroup or the tetrapod ingroup. The amphibian sequences form a clade separate from the human sequence. The bootstrap value for the amphibian clade was 100%. Within the amphibian clade the Bombina sequence was the sister group to a clade composed of the X. laevis and S. multiplicatus sequences. The bootstrap value for the X. laevis/S. multiplicatus clade was 94%. Collectively, these data indicate that the sequence of Bombina proenkephalin may be more similar to the proposed ancestral anuran proenkephalin sequence, than either X. laevis or S. multiplicatus proenkephalin.     

Organization of proenkephalin in amphibians: cloning of a proenkephalin cDNA from the brain of the anuran amphibian, Spea multiplicatus

Cloning of a proenkephalin cDNA from the pelobatid anuran amphibian, Spea multiplicatus, provides additional evidence that Leu-enkephalin, although present in the brain of anuran amphibians, is not encoded by the proenkephalin gene. The S. multiplicatus proenkephalin cDNA is 1375 nucleotides in length, and the open reading frame contains the sequences of seven opioid sequences. There are five copies of the Met-enkephalin sequence, as well as an octapeptide opioid sequence (YGGFMRNY) and a heptapeptide opioid sequence (YGGFMRF). In the proenkephalin sequence of S. multiplicatus the penultimate opioid is a Met-enkephalin sequence rather than the Leu-enkephalin present in mammalian sequences. The same order of opioid sequences also is observed for the proenkephalin sequence of the pipid anuran amphibian, Xenopus laevis. Hence, from a phylogenetic standpoint the organization of tetrapod proenkephalin has been remarkably conserved. What remains to be resolved is whether the Leu-enkephalin sequence found in mammalian proenkephalin is an ancestral trait or a derived trait for the tetrapods. Unlike the proenkephalin precursor of X. laevis, all of the opioid sequences in the S. multiplicatus proenkephalin cDNA are flanked by paired-basic amino acid proteolytic cleavage sites. In this regard the proenkephalin sequence for S. multiplicatus is more similar to mammalian proenkephalins than the proenkephalin sequence of X. laevis. However, a comparison of the proenkephalin sequences in human, X. laevis, and S. multiplicatus revealed several conserved features in the evolution of the tetrapod proenkephalin gene. By contrast, a comparison of tetrapod proenkephalin sequences with the partial sequence of a sturgeon proenkephalin cDNA indicates that the position occupied by the penultimate opioid sequence in vertebrate proenkephalins may be a highly variable locus in this gene.     

Presence of proenkephalin in chromaffin cells of the frog adrenal gland

Using a specific antiserum to bovine proenkephalin 1–77 (synenkephalin), the distribution of this peptide in the frog adrenal gland has been studied by means of the indirect immunofluorescence technique. Proenkephalin immunoreactivity was found in all chromaffin cells, which also demonstrated enkephalin- and vasoactive intestinal peptide-like immunoreactivity. No nerve endings containing proenkephalin-, enkephalin-, or vasoactive intestinal peptide-like material could be detected. These data suggest a precursor-product mode of biosynthesis for enkephalins in amphibian chromaffin cells. On a phylogenic point of view, they further indicate a high stability of the structure of proenkephalin during the evolution process.     

Cloning and expression in Escherichia coli of the synthetic proenkephalin analogue gene

Enkephalins are pentapeptides with opioid activity that have been found in brain and other neural tissues. They are released by proteolytic processing of the proenkephalin, which contains several enkephalin sequences each flanked by pairs of basic amino acid (aa) residues. We have constructed an artificial variant of the proenkephalin gene by concatenation of synthetic oligodeoxynucleotides (oligo) coding for Met-enkephalin preceded by two arginines. One of the resulting structures, containing eleven enkephalin sequences separated by pairs of arginine codons, was cloned in the expression vector pRE31. The biological activity of enkephalin was detected after the digestion of the isolated plasmid-coded protein with trypsin and carboxypeptidase B. The product of the synthetic gene may thus serve as a defined simplified substrate for the study of the not yet fully understood enzymatic mechanisms of proenkephalin processing.     

Cloning and partial nucleotide sequence of Schistosoma japonicum paramyosin: a potential vaccine candidate against schistosomiasis.

, , , and 1992. Cloning and partial nucleotide sequence of Schistosoma japonicum paramyosin: a potential vaccine candidate against schistosomiasis. International Journal for Parasitology 22: 1187–1191. Paramyosin from the blood fluke, Schistosoma mansoni, has shown promise as a vaccine candidate for schistosomiasis mansoni. Here we report the cloning and partial nucleotide sequence of a cDNA encoding paramyosin from the related human parasite, Schistosoma japonicum. Affinity purified antibodies to this clone recognized a S. japonicum antigen of molecular weight 97 kDa, equivalent to the reported size of S. mansoni paramyosin. Alignment of the cDNA sequence with that of S. mansoni paramyosin revealed 90% identity. Comparison of the predicted amino acid sequences revealed 95% identity. Although these two parasites differ in many characteristics, the substantial homology demonstrated here between S. mansoni and S. japonicum paramyosin could have important implications for the development of a S. japonicum vaccine.     

Cloning, sequencing, and expression of the meso-diaminopimelate dehydrogenase gene from Bacillus sphaericus

The gene encoding the meso-diaminopimelate dehydrogenase of Bacillus sphaericus was cloned into E. coli cells and its complete DNA sequence was determined. The meso-diaminopimelate dehydrogenase gene consisted of 978 nucleotides and encoded 326 amino acid residues corresponding to the subunit of the dimeric enzyme. The amino acid sequence deduced from the nucleotide sequence of the enzyme gene of B. sphaericus showed 50% identity with those of the enzymes from Corynebacterium glutamicum and Brevibacterium flavum. The enzyme gene from B. sphaericus was highly expressed in E. coli cells. We purified the enzyme to homogeneity from a transformant with 76% recovery. The N-terminal amino acid of both the enzyme from B. sphaericus and the transformant were serine, indicating that the N-terminal methionine is removed by post-translational modification in B. sphaericus and E. coli cells.     

The cDNA encoding Xenopus laevis heat-shock factor 1 (XHSF1): nucleotide and deduced amino-acid sequences, and properties of the encoded protein

We have isolated a cDNA encoding Xenopus laevis (Xl) heat-shock factor 1 (XHSF1). XHSF1, translated from the mRNA synthesized in vitro, will bind specifically to the X1 hsp70 promoter (hsp70). Microinjection of XHSF1 mRNA into Xl oocytes leads to synthesis of XHSF1 which accumulates in the nucleus and selectively activates Xl phsp70p activity at 18°C.     

Cladistic analysis of anuran POMC sequences

Procedures for performing cladistic analyses can provide powerful tools for understanding the evolution of neuropeptide and polypeptide hormone coding genes. These analyses can be done on either amino acid data sets or nucleotide data sets and can utilize several different algorithms that are dependent on distinct sets of operating assumptions and constraints. In some cases, the results of these analyses can be used to gauge phylogenetic relationships between taxa. Selecting the proper cladistic analysis strategy is dependent on the taxonomic level of analysis and the rate of evolution within the orthologous genes being evaluated. For example, previous studies have shown that the amino acid sequence of proopiomelanocortin (POMC), the common precursor for the melanocortins and beta-endorphin, can be used to resolve phylogenetic relationships at the class and order level. This study tested the hypothesis that POMC sequences could be used to resolve phylogenetic relationships at the family taxonomic level. Cladistic analyses were performed on amphibian POMC sequences characterized from the marine toad, Bufo marinus (family Bufonidae; this study), the spadefoot toad, Spea multiplicatus (family Pelobatidae), the African clawed frog, Xenopus laevis (family Pipidae) and the laughing frog, Rana ridibunda (family Ranidae). In these analyses the sequence of Australian lungfish POMC was used as the outgroup. The analyses were done at the amino acid level using the maximum parsimony algorithm and at the nucleotide level using the maximum likelihood algorithm. For the anuran POMC genes, analysis at the nucleotide level using the maximum likelihood algorithm generated a cladogram with higher bootstrap values than the maximum parsimony analysis of the POMC amino acid data set. For anuran POMC sequences, analysis of nucleotide sequences using the maximum likelihood algorithm would appear to be the preferred strategy for resolving phylogenetic relationships at the family taxonomic level.     

Searching for the closest living relative(s) of tetrapods through evolutionary analyses of mitochondrial and nuclear data

The phylogenetic relationships of the African lungfish (Protopterus dolloi) and the coelacanth (Latimeria chalumnae) with respect to tetrapods were analyzed using complete mitochondrial genome DNA sequences. A lungfish + coelancanth clade was favored by maximum parsimony (although this result is dependent on which transition:transversion weights are applied), and a lungfish + tetrapod clade was supported by neighbor-joining and maximum-likelihood analyses. These two hypotheses received the strongest statistical and bootstrap support to the exclusion of the third alternative, the coelacanth + tetrapod sister group relationship. All mitochondrial protein coding genes combined favor a lungfish + tetrapod grouping. We can confidently reject the hypothesis that the coelacanth is the closest living relative of tetrapods. When the complete mitochondrial sequence data were combined with nuclear 28S rRNA gene data, a lungfish + coelacanth clade was supported by maximum parsimony and maximum likelihood, but a lungfish + tetrapod clade was favored by neighbor-joining. The seeming conflicting results based on different data sets and phylogenetic methods were typically not statistically strongly supported based on Kishino-Hasegawa and Templeton tests, although they were often supported by strong bootstrap values. Differences in rate of evolution of the different mitochondrial genes (slowly evolving genes such as the cytochrome oxidase and tRNA genes favored a lungfish + coelacanth clade, whereas genes of relatively faster substitution rate, such as several NADH dehydrogenase genes, supported a lungfish + tetrapod grouping), as well as the rapid radiation of the lineages back in the Devonian, rather than base compositional biases among taxa seem to be directly responsible for the remaining uncertainty in accepting one of the two alternate hypotheses.     

Biochemistry of the enkephalins and enkephalin-containing peptides

The enkephalins are present in many tissues not only as the free pentapeptides, but also as internal sequences in larger polypeptides of varying size. Fourteen enkephalincontaining peptides (EC peptides) from beef adrenal medulla were isolated and sequenced, and the presence of a protein that contained several [Met]enkephalin sequences and one of [Leu]enkephalin was demonstrated. Because the latter was assumed to represent the gene product, it was named proenkephalin. Sequence data from the EC peptides made possible the synthesis of a polynucleotide probe with essentially no degeneracy and permitted the cloning of a partial proenkephalin cDNA. The complete structure of proenkephalin was deduced from both peptide and cDNA sequencing data. Proenkephalin is now known to be one of three enkephalin-containing gene products, each of which gives rise to many physiologically active peptides.     

Host reactions in Biomphalaria glabrata to Schistosoma mansoni miracidia, involving variations in parasite strains, numbers and sequence of exposures

M-line Biomphalaria glabrata snails are susceptible to Puerto Rican (PR-1) strain of Schistosoma mansoni, but are resistant to a St. Lucian (LC-1) strain. 10-R2 B. glabrata snails are resistant to both strains of S. mansoni. When 10-R2 snails were exposed repeatedly to PR-1 S. mansoni miracidia for 5 consecutive days, all of the sporocysts were encapsulated and destroyed by the snails. Thirty-four per cent of sporocysts examined in M-line snails with similar exposures were also degraded. In double concurrent infections of M-line B. glabrata with [³H]leucine-labeled and unlabeled PR-1 and Lc-1 S. mansoni, the incompatible Lc-1 miracidia were selectively attacked and destroyed. This destruction occurred irrespective of the sequence of exposure of the 2 strains of miracidia, and whether or not the miracidia were labeled. Successful superinfection of M-line B. glabrata with homologous S. mansoni miracidia was obtained at least 4 days after the primary exposure to the miracidia.     

Expression of the human proenkephalin gene in mouse pituitary cells: accurate and efficient mRNA production and proteolytic processing

A recombinant plasmid containing the human proenkephalin gene ligated to pBR322 was introduced into a mouse pituitary cell line (AtT-20D16v) that normally expresses pro-opiomelanocortin but not proenkephalin. The plasmid was introduced by co-transformation with the G418-selectable plasmid, pRSVneo. Stable transformants were isolated and analyzed for the presence of the human proenkephalin gene. AtT-20 transformants which had one or more copies of the human proenkephalin gene integrated stably into the mouse chromosomal DNA expressed a 1.45 kb mRNA identical in size to human proenkephalin mRNA. Primer extension analysis indicated that the human proenkephalin gene was accurately and efficiently transcribed from its own promoter. AtT-20 transformants that expressed the 1.45 kb human proenkephalin mRNA also expressed proenkephalin protein and cleaved the protein to form free Met-enkephalin. This is of particular interest because these cells do not cleave all of the available pairs of basic amino acids in the endogenous protein, pro-opiomelanocortin, the precursor to ACTH, beta-endorphin and melanocyte stimulating hormones. The release of both ACTH and Met-enkephalin from these cells is stimulated by corticotropin releasing factor, a natural secretagogue for ACTH, indicating that the two classes of peptide share a related secretory pathway.     

Sequences of two expressed sequence tags (EST) from rice encoding different cap-binding proteins

Wheat has been shown to have two forms of the cap-binding protein that participate in the initiation of translation. To identify cap-binding proteins from other higher plant species, the expressed sequence tag (EST) database was searched. Several rice ESTs were identified with similarity to both forms of the wheat cap-binding proteins. Two of the rice ESTs were obtained and the cDNA sequences completed. The deduced amino acid sequences of the rice cap-binding proteins are compared to the wheat cap-binding proteins and cap-binding proteins from Saccharomyces cerevisiae, Drosophila melanogaster, Xenopus laevis and human.     

植物提取物和药剂对蔬菜蚜虫种群的联合控制作用

研究白蝴蝶(Syngonium podophyllum)乙醇提取物、苍耳(Xanthium sibiricum)乙醇提取物、机油乳剂和0.3%印楝素乳油对桃蚜(Myzus persicae)和萝卜蚜(Lipaphis erysimi)的控制效果.室内四因子(1/2实施)二次正交回归旋转组合设计测试对有翅蚜的忌避作用,结果表明,对有翅桃蚜的主要忌避作用物为苍耳乙醇提取物,当苍耳提取物与白蝴蝶乙醇提取物混用,以及机油乳剂和印楝素混用时,对桃蚜有翅蚜的忌避效果提高;而对萝卜蚜有翅蚜的忌避作用主要受苍耳提取物和白蝴蝶提取物的影响.这些干扰作用均是非线性的.田间试验结果,单独使用白蝴蝶提取物对有翅成蚜有较强的驱避作用;对萝卜蚜自然种群的干扰控制作用以4种植物提取物和药剂混配效果最好,达95.7%;对桃蚜自然种群干扰控制效果最好的则是白蝴蝶提取物,控制效果达87%;同时,白蝴蝶提取物与印楝素乳油混配以及苍耳提取物与印楝素乳油混配,对两种蚜虫的控制效果均达80%以上.     

Purification from brain of synenkephalin, the N-terminal fragment of proenkephalin

Abstract: The primary sequence of adrenal proenkephalin was recently deduced from the structure of the cloned cDNA that codes for this protein. Several enkephalin-containing proteins with molecular weights between 8,000 and 20,000 daltons were purified from the bovine adrenal medulla. These proteins appear to represent intermediates in the processing of proenkephalin into physiologically active opioid peptides. While the concentrations of these large processing intermediates in the adrenal medulla are quite high, similar proteins have not yet been shown to be present in brain, and there is some question as to whether the brain synthesizes an enkephalin precursor similar to adrenal proenkephalin. We report here the purification from bovine caudate nucleus of synenkephalin, the N-terminal fragment of adrenal proenkephalin. The amino acid composition of synenkephalin indicates that the protein represents residues 1–70 of adrenal proenkephalin. Thus the brain and adrenal glands appear to utilize a similar precursor for enkephalin biosynthesis.