Ermelin, an endoplasmic reticulum transmembrane protein, contains the novel HELP domain conserved in eukaryotes

We have cloned a cDNA encoding a novel protein referred to as ermelin from mouse C2 skeletal muscle cells. This protein contained six hydrophobic amino acid stretches corresponding to transmembrane domains, two histidine-rich sequences, and a sequence homologous to the fusion peptides of certain fusion proteins. Ermelin also contained a novel modular sequence, designated as HELP domain, which was highly conserved among eukaryotes, from yeast to higher plants and animals. All these HELP domain-containing proteins, including mouse KE4, Drosophila Catsup, and Arabidopsis IAR1, possessed multipass transmembrane domains and histidine-rich sequences. Ermelin was predominantly expressed in brain and testis, and induced during neuronal differentiation of N1E-115 neuroblastoma cells but downregulated during myogenic differentiation of C2 cells. The mRNA was accumulated in hippocampus and cerebellum of brain and central areas of seminiferous tubules in testis. Epitope-tagging experiments located ermelin and KE4 to a network structure throughout the cytoplasm. Staining with the fluorescent dye DiOC(6)(3) identified this structure as the endoplasmic reticulum. These results suggest that at least some, if not all, of the HELP domain-containing proteins are multipass endoplasmic reticulum membrane proteins with functions conserved among eukaryotes.     

三疣梭子蟹NF-κB家族基因Relish和Dorsal的克隆及表达特征

为研究Relish和Dorsal在三疣梭子蟹免疫过程中所起到的作用, 研究利用RACE技术克隆获得三疣梭子蟹Relish(Pt-Rel)、Dorsal基因(Pt-Dor) cDNA全长, 并通过实时荧光定量PCR技术分析了Pt-Rel和Pt-Dor基因在健康蟹不同组织及其原代培养的血淋巴细胞在感染不同微生物后的表达情况。结果显示, Pt-Rel cDNA长3254 bp, ORF长2949 bp, 编码983个氨基酸, Pt-Dor cDNA长2348 bp, ORF长1911 bp, 编码637个氨基酸; 蛋白结构预测分析发现Pt-Rel和Pt-Dor均包含RHD (Rel homology domain)及IPT (Immunoglobulin-like fold, Plexins, TranscriPtion factors)Rel/NF-κB家族蛋白经典结构域。Pt-Rel和Pt-Dor与其他节肢动物Relish、Dorsal氨基酸序列具有很高的相似性; 在系统进化分析中Pt-Rel和Pt-Dor分别与中华绒螯蟹等甲壳动物的Relish、Dorsal聚在一支, 而昆虫类聚在另一支。Pt-Rel和Pt-Dor在检测的6种组织中均有表达, 且2个基因均在血淋巴细胞中表达量最高。蟹血淋巴细胞体外感染实验结果表明, 不同病原微生物对2个基因表达的影响非常相似, 假丝酵母在2h明显诱导了Pt-Rel和Pt-Dor基因的表达, 而金黄色葡萄球菌及溶藻弧菌在感染4h后使Pt-Rel和Pt-Dor基因的表达显著上调。上述研究结果表明Pt-Rel和Pt-Dor基因很有可能参与了三疣梭子蟹的抗感染免疫过程。     

The chicken IL-1 receptor: differential evolution of the cytoplasmic and extracellular domains.

The evolutionary conservation of a sequence or part of it can help to identify the essential functional and structural domains within a protein. We have cloned and characterised a cDNA coding for the type-I interleukin-1 receptor (IL-1R) of chick (ch) embryo fibroblasts. The comparison of the amino acid (aa) sequences of the avian with that of murine (m) and human (h) IL-1Rs shows a 60% homology. The intracellular domain is the most conserved region of the chIL-1R, showing 76-79% homology to the murine and human sequences, respectively. The striking conservation of the cytoplasmic region of the receptor is confirmed by its homology with the Toll receptor protein of Drosophila melanogaster. The alignment between the chicken and D. melanogaster proteins shows the presence of four aa blocks with more than 80% homology. The possible functional significance of this homology is discussed. The extracellular binding region of the receptor has a clearly recognisable immunoglobulin-like structure although the sequence divergence is higher than in the cytoplasmic domain.     

REC, a new member of the MCM-related protein family, is required for meiotic recombination in Drosophila

rec mutations result in an extremely low level of recombination and a high frequency of primary non-disjunction in the female meiosis of Drosophila melanogaster. Here we demonstrate that the rec gene encodes a novel protein related to the mini-chromosome maintenance (MCM) proteins. Six MCM proteins (MCM2-7) are conserved in eukaryotic genomes, and they function as heterohexamers in the initiation and progression of mitotic DNA replication. Three rec alleles, rec(1), rec(2) and rec (3), were found to possess mutations within this gene, and P element-mediated germline transformation with a wild-type rec cDNA fully rescued the rec mutant phenotypes. The 885 amino acid REC protein has an MCM domain in the middle of its sequence and, like MCM2, 4, 6 and 7, REC contains a putative Zn-finger motif. Phylogenetic analyses revealed that REC is distantly related to the six conserved MCM proteins. Database searches reveal that there are candidates for orthologs of REC in other higher eukaryotes, including human. We addressed whether rec is involved in DNA repair in the mitotic division after the DNA damage caused by methylmethane sulfonate (MMS) or by X-rays. These analyses suggest that the rec gene has no, or only a minor, role in DNA repair and recombination in somatic cells.     

Cloning of the human cDNA which can complement the defect of the yeast mannosyltransferase I-deficient mutant alg 1

The assembly of the lipid-linked oligosaccharide, Glc(3)Man(9)GlcNAc(2)-P-P-Dol, occurs on the rough ER membrane in an ordered stepwise manner. The process is highly conserved among eukaryotes. In order to isolate the human mannosyltransferase I (MT-I) gene involved in the process, we used the Saccharomyces cerevisiae MT-I gene ( ALG1 ), which has already been cloned. On searching the EST database with the amino acid sequence of the ALG1 gene product, we detected seven related human EST clones. A human fetal brain cDNA library was screened by PCR using gene-specific primers based on the EST nucleotide sequences and a 430 bp cDNA fragment was amplified. The cDNA library was rescreened with this 430 bp cDNA, and two cDNA clones (HR1-3 and HR1-4) were isolated and sequenced. On a homology search of the EST database with the nucleotide sequence of HR1-3, we detected a novel human EST clone, AA675921 (GenBank accession number). Based on the nucleotide sequences of AA675921 and HR1-4, we designed gene-specific PCR primers, which allowed to amplify a 1.8 kb cDNA from human fetal brain cDNA. This cDNA was cloned and shown to contain an ORF encoding a protein of 464 amino acids. We designated this ORF as Hmat-1. The amino acid sequence deduced from the Hmat-1 gene showed several highly conserved regions shared with the yeast and nematode MT-I sequences. Furthermore, this 1.8 kb cDNA successfully complemented the S. cerevisiae alg1-1 mutation, indicating that the Hmat-1 gene encodes the human MT-I and that the function of this enzyme was conserved between yeast and human.     

Isolation and characterization of a gene encoding DNA topoisomerase I in Drosophila melanogaster.

We synthesized a DNA probe specific for the gene encoding eucaryotic DNA topoisomerase I by the polymerase chain reaction. The sequences of the primers for this reaction were deduced from the regions with extensive homology among the enzymes from the fission and budding yeasts, and the human. From the clones isolated by screening a Drosophila cDNA library with this DNA probe, two cDNA clones of 3.8 and 5.2 kb were characterized and completely sequenced. Both cDNA sequences contain an identical open reading frame for 972 amino acid residues. The 3.8 kb messenger RNA is likely generated by using a polyadenylation site 5' upstream to that used in generating the 5.2 kb mRNA. The predicted amino acid sequence shows that a segment of 420 amino acid residues at the amino terminus is hydrophilic, similar to the amino terminal 200 residues in the yeast and human enzymes. Furthermore, the Drosophila enzyme is unique in that the amino terminal 200 residues are enriched in serine and histidine residues; most of them are present in clusters. The rest of the Drosophila sequence is highly homologous to those from yeast and human enzymes. The evolutionarily conserved residues are identified and are likely the critical elements for the structure and function of this enzyme. A plasmid vector containing the cloned cDNA was constructed for the expression of Drosophila protein in Escherichia coli. The enzymatic and immunochemical analysis of the polypeptide produced in this heterologous expression system demonstrated that the expressed protein shares similar enzymatic properties and antigenic epitopes with DNA topoisomerase I purified from Drosophila embryos or tissue culture cells, thus establishing the bacterial expression system being useful for the future structure/function analysis of the Drosophila enzyme.     

A novel type of RNase III family proteins in eukaryotes

The RNase III family of double-stranded RNA-specific endonucleases is characterized by the presence of a highly conserved 9 amino acid stretch in their catalytic center known as the RNase III signature motif. We isolated the drosha gene, a new member of this family in Drosophila melanogaster. Characterization of this gene revealed the presence of two RNase III signature motifs in its sequence that may indicate that it is capable of forming an active catalytic center as a monomer. The drosha protein also contains an 825 amino acid N-terminus with an unknown function. A search for the known homologues of the drosha protein revealed that it has a similarity to two adjacent annotated genes identified during C. elegans genome sequencing. Analysis of the genomic region of these genes by the Fgenesh program and sequencing of the EST cDNA clone derived from it revealed that this region encodes only one gene. This newly identified gene in nematode genome shares a high similarity to Drosophila drosha throughout its entire protein sequence. A potential drosha homologue is also found among the deposited human cDNA sequences. A comparison of these drosha proteins to other members of the RNase III family indicates that they form a new group of proteins within this family.     

Molecular cloning and characterization of rat estrogen receptor cDNA.

A cDNA clone of rat uterus estrogen receptor (ER) has been isolated and sequenced. This clone contains a complete open reading frame encoding 600 amino acid residues which is 5 and 11 amino acids larger than the corresponding molecules of human and chicken, respectively. The molecular weight of this protein is calculated to be 67,029. When this clone was ligated to the pSV2 vector and transfected into COS7 cells, a protein was produced that had the same affinity to estrogen as rat uterus ER. This sequence shows 88% homology with human ER; 528 amino acids are identical and 14 amino acids are conservative substitutions. The comparison of rat, human and chicken ER sequences indicate the presence of three highly conserved regions suggesting that these regions play important roles in ER function. The putative DNA-binding domain is completely identical in rat, human and chicken. The C-terminal half region which is thought to be the estrogen binding domain is also highly conserved and is rich in hydrophobic amino acid residues. Southern blot analysis of genomic DNA with ER cDNA as a probe has shown that related sequences are present in the genome.     

Heat-shock proteins, Hsp84 and Hsp86, of mice and men: two related genes encode formerly identified tumour-specific transplantation antigens

Mouse cDNA clones have been isolated with the help of Drosophila melanogaster 82-kDa heat-shock protein (Hsp82)-coding sequences as hybridization probe. Sequencing of the overlapping mouse clones reveals a long open reading frame (ORF) that encodes a polypeptide of 83.3 kDa which shows about 80% similarity to the respective Drosophila Hsp82 amino acid sequence. The N-terminal half of this cDNA cross-hybridizes to a different class of mouse cDNA clones indicating a related gene. Northern blot hybridization experiments reveal a 2.6-kb poly(A)+RNA when probed with the hsp84 clone and a 2.85-kb signal with the hsp84-related cDNA. The amino acid sequences deduced from the contiguous ORF of the hsp84 and the hsp84-related cDNA coincide with the N-terminal sequence of formerly identified 84-kDa and 86-kDa tumour-specific transplantation antigens (Ullrich et al., 1986). In addition, the amino acid composition of the putative 84-kDa mouse Hsp described here is very similar to that of the 84-kDa tumour antigen described by Ullrich et al. (1986). Both observations corroborate the assumption that these Hsps are identical to the described 84-kDa and 86-kDa tumour-specific transplantation antigens. Using these mouse hsp gene clones as hybridization probes we also isolated the corresponding pair of human cDNA clones. Comparison of the respective sequences reveals a strong evolutionary constraint on these two genes in mouse and man.     

Sequence of a cDNA encoding turtle high mobility group 1 protein

In order to understand sequence information about turtle HMG1 gene, a cDNA encoding HMG1 protein of the Chinese soft-shell turtle (Pelodiscus sinensis) was amplified by RT-PCR from kidney total RNA, and was cloned, sequenced and analyzed. The results revealed that the open reading frame (ORF) of turtle HMG1 cDNA is 606 bp long. The ORF codifies 202 amino acid residues, from which two DNA-binding domains and one polyacidic region are derived. The DNA-binding domains share higher amino acid identity with homologues sequences of chicken (96.5%) and mammalian (74%) than homologues sequence of rainbow trout (67%). The polyacidic region shows 84.6% amino acid homology with the equivalent region of chicken HMG1 cDNA. Turtle HMG1 protein contains 3 Cys residues located at completely conserved positions. Conservation in sequence and structure suggests that the functions of turtle HMG1 cDNA may be highly conserved during evolution. To our knowledge, this is the first report of HMG1 cDNA sequence in any reptilian.     

Identification and characterization of a gene encoding a kinesin-like protein in Drosophila

We identified and sequenced a cDNA clone encoding a kinesin-like protein from Drosophila. The predicted product of this cDNA has a carboxy-terminal domain that is substantially similar to the motor domain of kinesin heavy chain. The amino-terminal domain is unlike that found in previously identified kinesins or kinesin-like proteins. Analyses of this new sequence suggest that the maximal motor unit in the kinesin superfamily may be as little as 350 amino acids, and that the existence of both kinesin and kinesin-like molecules must be an evolutionarily ancient feature of eukaryotes. We also tested some of the biochemical properties of the protein encoded by this cDNA and found them to be similar to those of kinesin. Finally, the clone we isolated appears to correspond to the non-claret disjunctional (ncd) gene, which when mutant causes defects in meiotic and early embryonic mitotic chromosome segregation, and whose recently determined sequence predicts a kinesin-like domain.     

Evolutionary conservation of recombination proteins and variability of meiosis-specific proteins of chromosomes

A comparison of amino acid sequences is performed for orthologs to the meiosis-specific proteins in humans and seven other species, including animals, fungi, and plants that serve as models for the study of molecular mechanisms of meiosis. It is demonstrated that the RAD51 recombination mediator protein is the most conserved of the studied proteins. Its meiotic homolog DMC1 is less conserved, like the MHL1 mismatch-repair protein. The meiosis-specific SPO11 endonuclease is the least conserved among the studied meiotic enzymes. Structural proteins of meiotic chromosomes are poorly conserved. REC8 meiotic cohesin has 6 times lower similarity in the organisms from different kingdoms than its somatic homolog RAD21. The intermediate conservation level is characteristic of the synaptonemal complex proteins containing HORMA domain. Two functional domains of SPO11 endonuclease and MutL Trans_MLH1 domain of MLH1 enzyme are equally or even less conserved than the whole proteins. HORMA functional domain of a number of synaptonemal complex proteins is only 2–3 times more conserved than the whole molecule. Thus, among the key meiotic proteins, the most conserved are proteins responsible for the accuracy of meiotic recombination. Cohesins, synaptonemal complex proteins, and meiosis-specific SPO11 endonuclease are less conserved even within their functional domains. Obviously, the meiosis-specific proteins have undergone independent evolution in different phylogenetic lineages of eukaryotes.     

巴西橡胶树磷脂酰肌醇转移蛋白cDNA的克隆及其序列分析

本文从巴西橡胶树(Hevea brasiliensis)差减cDNA文库中筛选到一个与磷脂酰肌醇转移蛋白(phos-phatidylinositol transfer protein)同源性较高的基因片段,并根据该基因片段序列信息,设计特异性引物,采用cDNA末端快速扩增技术RACE(rapid amplification of cDNA ends)进行差异片段的5'和3'端的扩增,并获得长度为1081bp的全长cDNA克隆R291(GenBank登陆号:AY589690)。序列分析表明,该基因包含702bp的开放阅读框,编码234个氨基酸,推测其蛋白质的分子量为26.8kD,等电点为6.51,有一个的跨膜螺旋区(氨基酸位点为83～103)。R291基因含有一个脂质结合保守区(Sec14p-like lipid-binding domain),具有CRAL-TRIO脂质结合结构域,推测该基因是一个磷脂酰肌醇转移蛋白基因。该基因的克隆将为橡胶树磷脂酰肌醇代谢的研究奠定了基础,将有助于进一步了解磷脂酰肌醇代谢与胶乳再生之间的关系。     

毕氏海蓬子SbDREB基因的克隆与表达分析研究

以毕氏海蓬子的基因组为模板,通过PCR技术扩增到一个编码DREB蛋白AP2保守结构域的基因片段;根据该片段序列设计引物,以毕氏海蓬子经NaCl处理的植株肉质茎cDNA为模板,应用RACE技术获得该基因的cDNA全长,命名为SbDREB(GenBank登录号:JF894301)。SbDREB基因cDNA全长1206bp,包含一个编码284个氨基酸的完整开放阅读框。对氨基酸序列比对分析表明,该蛋白在靠近N端具有典型的AP2/EREBP保守结构域,且该结构域与一些高等植物DREB类转录因子的AP2区域具有高度同源性。进化树分析表明SbDREB属于DREB亚家族中的A-6亚族。实时荧光定量PCR结果显示:干旱、高盐和ABA能够诱导其表达,而低温则使其表达下调,表明该基因在毕氏海蓬子植株对干旱、盐和低温等非生物胁迫的应答中起作用。