斜带石斑鱼生长激素/催乳素家族基因cDNAs的分子克隆及其进化意义

从斜带石斑鱼垂体提取总。RNA,再取其50ng合成SMART cDNA。从所构建的垂体SMART cDNA质粒文库中筛选到生长激素／催乳素基因家族的2个成员的全长cDNA片段：生长激素(GH)基因全长为938bp,编码204个氨基酸;催乳素基因(PRI．)全长为1429bp,编码212个氨基酸。采用计算机软件Mega 2和CLUSTAL W1．64b对9种鱼的生长激素／催乳素基因家族的3个成员(GH、PRL和生长催乳素SL)的氨基酸序列进行系统分析,构建NJ分支系统树,对于序列中的插入／缺失位点则采用Pairaise Deletion,1000次自展(Bootstrap)分析计算各节点支持率。根据3个基因的氨基酸序列构建的系统树表明,石斑鱼与金头鲷、金鲈和牙鲆聚成一类,虹鳟与大马哈鱼聚成一类,鲫鱼与鲶鱼聚成一类,鳗鲡成另外一类。根据石斑鱼全长cDNA推断的氨基酸序列比较表明,SL相对GH和PRL有较高的保守性。石斑鱼的GH、PRL和SL的氨基酸同源性在24％～31％,但其C-端的氨基酸同源性较高,尤其是C-端的3个Cys是严格保守的。其中SL与GH的同源性(30．8％)高于与PRL的同源性(25．6％),GH和PRL的同源性最低(24．1％)。     

Cloning and bioinformatic analysis of an acidophilic β-mannanase gene, Anman5A, from Aspergillus niger LW-1

Using 3′ and 5′ rapid amplification of cDNA ends (RACE) techniques, the full-length cDNA sequence of the Anman5A, a gene that encodes an acidophilic β-mannanase of Aspergillus niger LW-1 (abbreviated to AnMan5A), was identified from the total RNA. The cDNA sequence was 1417 bp in length, harboring 5′- and 3′-untranslated regions, as well as an open reading frame (ORF) which encodes a 21-aa signal peptide, a 17-aa propeptide and a 345-aa mature peptide. Based on the topology of the phylogenetic tree of β-mannanases from glycoside hydrolase (GH) family 5, the AnMan5A belongs to the subfamily 7 of the GH family 5. Its 3-D structure was modeled by the bitemplate-based method using both MODELLER 9.9 and SALIGN programs, based on the known β-mannanase crystal structures of Trichoderma reesei (1QNO) and Lycopersicon esculentum (1RH9) from the GH family 5. In addition, the complete DNA sequence of the Anman5A was amplified from the genomic DNA using the pUCm-T vector-mediated PCR and conventional PCR methods. The DNA sequence was 1825 bp in length, containing a 5′-flanking regulatory region, 2 introns and 3 exons when compared with the full-length cDNA.     

Cloning and sequencing of cDNA encoding the cat growth hormone

A cDNA encoding cat growth hormone (Fc) GH) has been isolated and sequenced. This is the first report of a feline GH nucleotide and deduced amino acid (aa) sequences. This cat pituitary cDNA resembles a typical mammalian pre-GH cDNA with its encoded mature hormone differing from dog GH only by a single as residue.     

Carp growth hormone: molecular cloning and sequencing of cDNA

cDNA clones of the fish Cyprinus carpio growth hormone (GH) mRNA have been isolated from a cDNA library prepared from carp pituitary gland poly(A)+RNA. The nucleotide sequence of one of the carp GH cDNA clones containing an insert of 1164 nucleotides (nt) was determined. The cDNA sequence was found to encode a polypeptide of 210 amino acids (aa) including a signal peptide of 22 aa and to contain 5' and 3' untranslated regions of the mRNA of 36 and 498 nt, respectively. The carp GH presents a 63% amino acid sequence homology with the salmon GH, has structural features common with other GH polypeptides of mammalian or avian origin and contains domains of conserved sequence near the N- and C-terminal regions. Southern blot hybridization of carp genomic DNA with GH cDNA probes shows the presence of at least two GH-coding sequences in the fish genome.     

The cDNA sequence of murine Nkx-2.2

We isolated and sequenced a 2026-bp murine Nkx-2.2 cDNA clone that contains an open reading frame encoding 273 amino acids (aa). The 273-aa protein includes a homeobox, an NK-2 box and an N-terminal decapeptide found in other NK family members.     

Identification of a cDNA encoding a long form of prolactin receptor in human hepatoma and breast cancer cells

Human PRL receptor cDNA clones from hepatoma (Hep G2) and breast cancer (T-47D) libraries were isolated by using a rat PRL receptor cDNA probe. The nucleotide sequence predicts a mature protein of 598 amino acids with a much longer cytoplasmic domain than the rat liver PRL receptor. Although this extended region has additional segments of localized sequence identity with the human GH receptor, there is no identity with any consensus sequences known to be involved in hormonal signal transduction. This cDNA will be a valuable tool to better understand the role of PRL in the development and growth of human breast cancer.     

Exchanges of short polymorphic DNA segments predating speciation in feline major histocompatibility complex class I genes

Sequence comparisons of 14 distinct MHC class I cDNA clones isolated from species representing the three major taxonomic lineages of Felidae (domestic cat lineage, ocelot lineage, and pantherine lineage) revealed that feline MHC class I alleles have highly mosaic structures with short polymorphic sequence motifs that are rearranged between alleles of individual MHC loci, between MHC class I genes within cat species, and between homologous MHC loci in different species. The pattern of sequence variation in felids supports the role of the following factors in production and maintenance of MHC variation: (1) gradual spontaneous mutation; (2) selective pressure to conserve certain residues but also to vary in hypervariable regions, notably residues that functionally participate in antigen recognition and presentation; and (3) recombination-mediated gene exchange between alleles and between related genes. The overall amount of genetic variation observed among MHC class I genes in the Felidae family is no greater than the amount of variation within any outbred cat species (i.e., domestic cat, ocelot). The occurrence of equivalent levels of polymorphism plus the simultaneous persistence of the same sequence motifs in divergent feline species suggest that most MHC class I nucleotide site polymorphism predated species divergences. Ancient polymorphisms have been transmitted through the speciation events and modern feline MHC class I alleles were derived by recombinational exchange of polymorphic sequence motifs. Moreover, some of these sequence motifs were found in other mammalian MHC class I genes, such as classical human HLA-B5, nonclassical human HLA-E class I genes, and bovine class I genes. These results raise the prospect of an ancient origin for some motifs, although the possibility of convergence in parallel mammalian radiations cannot be excluded. Correspondence to: N. Yuhki     

Cloning of novel cellulases from cellulolytic fungi: Heterologous expression of a family 5 glycoside hydrolase from Trametes versicolor in Pichia pastoris

Total cDNA isolated from cellulolytic fungi cultured in cellulose was examined for the presence of sequences encoding for endoglucanases. Novel sequences encoding for glycoside hydrolases (GHs) were identified in Fusarium oxysporum, Ganoderma applanatum and Trametes versicolor. The cDNA encoding for partial sequences of GH family 61 cellulases from F. oxysporum and G. applanatum shares 58 and 68% identity with endoglucanases from Glomerella graminicola and Laccaria bicolor, respectively. A new GH family 5 endoglucanase from T. versicolor was also identified. The cDNA encoding for the mature protein was completely sequenced. This enzyme shares 96% identity with Trametes hirsuta endoglucanase and 22% with Trichoderma reesei endoglucanase II (EGII). The enzyme, named TvEG, has N-terminal family 1 carbohydrate binding module (CBM1). The full length cDNA was cloned into the pPICZαB vector and expressed as an active, extracellular enzyme in the methylotrophic yeast Pichia pastoris. Preliminary studies suggest that T. versicolor could be useful for lignocellulose degradation.     

Molecular Characterisation of a Novel Isoform of Hepatic Antimicrobial Peptide,Hepcidin (<Emphasis Type="Italic">Le</Emphasis>-Hepc), from <Emphasis Type="Italic">Leiognathus equulus</Emphasis> and Analysis of Its Functional Properties In Silico

Hepcidin represents a family of cysteine-rich antimicrobial peptides that are mainly expressed in the liver of living organisms. In this study, we have identified and characterised a novel isoform of hepcidin from the common pony fish, Leiognathus equulus (Le-Hepc). A 261-bp fragment cDNA coding for 86 amino acids was obtained. Homologous analysis showed that Le-Hepc belongs to the hepcidin super family and shares sequence identity with other known fish pre-propeptide hepcidin sequences. The ORF encodes for a 24-amino acid (aa) signal peptide coupled to a 36-aa prodomain followed by a 26-aa mature peptide. The mature peptide region has a calculated molecular weight of 2.73 kDa, a net positive charge of +2 and a theoretical pI of 8.23. Phylogenetic analysis of Le-Hepc showed a strong relationship with other fish hepcidin sequences and clustered into HAMP2 group hepcidins. Secondary structural analysis indicated that Le-Hepc mature peptide contains two antiparallel β-sheets strengthened by four disulphide bonds formed by eight conserved cysteine residues. The physicochemical properties of the peptide and its structural parameters are in agreement with characteristic features of an antimicrobial peptide. This is the first report of an antimicrobial peptide from the common pony fish, L. equulus.     

Cloning and analysis of a cDNA encoding a two-domain hemoglobin chain from the water flea Daphnia magna

A cDNA encoding a two-domain hemoglobin (Hb) chain of Daphnia magna was cloned and its nucleotide (nt) sequence of 1261 bp was determined. The nt sequence contained 74 bp of the leader sequence, 1047 bp of an open reading frame (ORF), and 119 bp of the 3′-untranslated region (UTR), excluding the polyadenylation tail. A sequence, AATACA, located 24 bp upstream from the polyA sequence was considered to be a polyadenylation signal. cDNA-derived amino acid (aa) sequence revealed that D. magna Hb chain is synthesized as a secretory precursor with a signal peptide of 18 aa. Mature D. magna Hb chain consists of 330-aa residues with a calculated molecular weight of 36 227, which is composed of two large repeated domains, domain 1 and 2. Several key aa that are invariant in all or most of other Hb and required for functional heme-binding are conserved in each of the two domains. The N-terminal extension (pre-A segment) of domain 1 was unusually long and contained an unusual threonine-rich sequence. The homology between the aa sequences of the two domains (24% identity) was much lower than that observed in other two-domain Hb chains from clams or nematode. Hb mRNA level in D. magna reared under low oxygen concentration was more than 12 times higher than that in D. magna reared with sufficient aeration, indicating that the expression of Hb gene is regulated by mRNA level.     

Discovery of a Novel Prolactin in Non-Mammalian Vertebrates: Evolutionary Perspectives and Its Involvement in Teleost Retina Development

Background

The three pituitary hormones, viz. prolactin (PRL), growth hormone (GH) and somatolactin (SL), together with the mammalian placental lactogen (PL), constitute a gene family of hormones with similar gene structure and encoded protein sequences. These hormones are believed to have evolved from a common ancestral gene through several rounds of gene duplication and subsequent divergence.

Principal Findings

In this study, we have identified a new PRL-like gene in non-mammalian vertebrates through bioinformatics and molecular cloning means. Phylogenetic analyses showed that this novel protein is homologous to the previously identified PRL. A receptor transactivation assay further showed that this novel protein could bind to PRL receptor to trigger the downstream post-receptor event, indicating that it is biologically active. In view of its close phylogenetic relationship with PRL and also its ability to activate PRL receptor, we name it as PRL2 and the previously identified PRL as PRL1. All the newly discovered PRL2 sequences possess three conserved disulfide linkages with the exception of the shark PRL2 which has only two. In sharp contrast to the classical PRL1 which is predominantly expressed in the pituitary, PRL2 was found to be mainly expressed in the eye and brain of the zebrafish but not in the pituitary. A largely reduced inner nuclear layer of the retina was observed after morpholino knockdown of zebrafish PRL2, indicating its role on retina development in teleost.

Significance

The discovery of this novel PRL has revitalized our understanding on the evolution of the GH/PRL/SL/PL gene family. Its unique expression and functions in the zebrafish eye also provide a new avenue of research on the neuroendocrine control of retina development in vertebrates.     

Cloning and expression of ovine placental lactogen

Ovine placental lactogen (oPL) is active in a wide range of GH and PRL assays, a property that it shares with human GH (hGH). In addition, oPL is one of a small number of hormones that bind the human GH receptor with high affinity. In order to compare the sequence of oPL to the sequences of other members of the GH family, full-length cDNA clones have been isolated. These clones predict that the full sequence of oPL contains 198 amino acids preceded by a 38 amino acid signal sequence. The mature oPL sequence includes six cysteine and two tryptophan residues and shows substantially more identity to bovine PL (67%) and oPL (49%) than to mouse (31%) or human (25%) PL or to oGH (28%) or (26%) hGH. Like the natural hormone, oPL expressed in mammalian tissue cells binds with high affinity to a soluble form of the recombinant hGH receptor. Thus, oPL binds to the human receptor in spite of having a sequence that is considerably divergent from hGH. Interestingly, the sequence of oPL differs from hGH at most of the amino acids recently found by mutagenesis studies to be important residues in the binding of hGH to the human receptor.     

Cloning and characterization of two processed pseudogenes and the cDNA for the murine U1 snRNP-specific protein C

Genes for the snRNP proteins U1-70K, U1-A, Sm-B′/B, Sm-D1 and Sm-E have been isolated from various metazoan species. The genes for Sm-D1 and Sm-E, which were isolated from a murine and human source respectively, appear to belong to a multigene family. It has been suggested that also for the mammalian U1-C protein such a multigene family exists. With the human U1-C cDNA as a probe, two genes containing sequences homologous to the probe sequence were isolated from a mouse genomic library. Simultaneously, a murine U1-C cDNA was isolated from a mouse cDNA library. This 0.74 kb cDNA contains an open reading frame (ORF) of 477 bp encoding a polypeptide of 159 amino acids (aa) which differs at only one position (position 65) from the human U1-C protein. One of the isolated U1-C genes contains an ORF as well and shares 92% nucleotide sequence identity with the mouse U1-C cDNA. The features of this gene, in particular the absence of introns, the acquisition of a 3′ poly(A) tail and flanking direct repeats, indicate that it represents a processed pseudogene. At the predicted aa sequence level, substitutions of conserved residues at functionally important positions are observed, strongly suggesting that expression of this gene would not lead to a functional polypeptide. The second U1-C gene appeared to be a pseudogene as well because it is also intronless and contains a frameshift mutation compared to the ORF in the mouse U1-C cDNA. The characterization of these two pseudogenes points to the existence of a U1-C multigene family in mice. Furthermore, comparison of aa sequences of the murine, human and Xenopus U1-C shows that the protein is highly conserved through evolution. Since the Xenopus U1-C differs from the two mammalian counterparts solely at a number of positions in the C-terminal region, it can be concluded that aa changes are less well tolerated in the N-terminal region of U1-C than in the rest of the protein.     

Cloning and characterization of ribonuclease T2 gene (RNHe30) from the basidiomycete,Hericium erinaceum

A gene encoding a ribonuclease T2 (RNase T2) family enzyme, RNHe30, was cloned from Hericium erinaceum by PCR. The deduced amino acid sequence from the complimentary DNA (cDNA) (1074 bp) encodes a 302-aa protein (RNase He30) that has the consensus amino acid sequences of RNase T2 family enzymes including the putative signal peptide. The presence of five introns in the genomic DNA was confirmed by comparison of the cDNA and genomic DNA sequences. The promoter region contains a putative CAAT box and a consensus TATA box. Genes coding homologous enzymes were also identified in various other basidiomycetes. A phylogenetic tree of RNase T2s from these fungi was constructed from a multiple alignment of the deduced amino acid sequences. The tree showed that the enzymes were divided into two main groups.