Cloning and partial characterization of a gene in Bombyx mori homologous to a human adiponectin receptor

In this study, we report the cloning and characteristics of an adiponectin-like receptor gene from Bombyx mori (BmAdipoR) with highly conserved deduced amino-acid sequences and similar structure to the human adiponectin receptor (AdipoR). Structural analysis of the translated cDNA suggested it encoded a membrane protein with seven transmembrane domains. BmAdipoR was found to be expressed in multiple tissues and highly expressed in Malpighian tubules, fat body and testis. BmNPV (Bombyx mori nucleopolyhedrovirus) bacmid system combined with confocal microscopy revealed that BmAdipoR was targeted to the cell membrane. We also found that infection with BmNPV did not have an effect on BmAdipoR mRNA quantity in the midgut of susceptible Bombyx mori strain (306) at 48 h, but BmAdipoR mRNA quantity increased significantly at 72 h. We concluded that BmAdipoR gene was a membrane protein ubiquitously expressed in Bombyx mori tissues and that its expression was altered by treating with BmNPV.     

Cloning and characterization of a Bacillus subtilis gene homologous to E. coli secY

A 3.5-kb HindIII DNA fragment containing the secY gene of Bacillus subtilis has been cloned into plasmid pUC13 using the Escherichia coli secY gene as a probe. The complete nucleotide sequence of the cloned DNA indicated that it contained five open reading frames, and their order in the region, given by the gene product, was suggested to be L30-L15-SecY-Adk-Map by their similarity to the products of the E. coli genes. The region was similar to a part of the spc operon of the E. coli chromosome, although the genes for Adk and Map were not included. The gene product of the B. subtilis secY homologue was composed of 423 amino acids and its molecular weight was calculated to be 46,300. The distribution of hydrophobic amino acids in the gene product suggested that the protein is a membrane integrated protein with ten transmembrane segments. The total deduced amino acid sequence of the B. subtilis SecY homologue shows 41.3% homology with that of E. coli SecY, but remarkably higher homologous regions (more than 80% identity) are present in the four cytoplasmic domains.     

Cloning and characterization of a gene encoding ABP57, a soluble auxin-binding protein

Auxin-binding protein 57 (ABP₅₇), a soluble auxin-binding protein, acts as a receptor to activate plasma membrane (PM) H⁺-ATPase. Here, we report the cloning of abp57 and the biochemical characterization of its protein expressed in E. coli. The analysis of internal amino acid sequences of ABP₅₇ purified from rice shoots enabled us to search for the corresponding gene in protein DB of NCBI. Further BLAST analysis showed that rice has four abp57-like genes and maize has at least one homolog. Interestingly, Arabidopsis seems to have no homolog. Recombinant ABP₅₇ expressed in E. coli caused the activation of PM H⁺-ATPase regardless of the existence of IAA. Scatchard analysis showed that the recombinant protein has relatively low affinity to IAA as compared to natural ABP₅₇. These results collectively support the notion that the cloned gene is responsible for ABP₅₇.     

Cloning and characterization of a third type of human alpha-amylase gene, AMY2B

We have previously reported concerning the existence of a third type of human alpha-amylase gene, AMY3 [Emi et al., Gene 62 (1988) 229-235; Tomita et al., Gene 76 (1989) 11-18], which is expressed in a lung carcinoid tissue, and differs in nucleotide sequence from the two previously characterized human alpha-amylase genes coding for salivary and pancreatic isozymes, termed AMY1 and AMY2, respectively. Here, we rename this gene AMY2B to coincide with the designation by Gumucio et al. [Mol. Cell Biol. 8 (1988) 1197-1205] and describe its genetic properties as revealed by sequencing studies. It consists of ten major exons whose sequences are highly homologous to those of AMY1 and AMY2. Not only the exons, but also most of the introns seem to be highly conserved, as judged from physical mapping data. The AMY2B gene identified from mRNA in a lung carcinoid tissue has at least two additional untranslated exons in its 5' region; hence the promoter lies far upstream relative to the other two AMY genes.     

Cloning and characterization of a bamboo LEAFY HULL STERILE1 homologous gene.

A cDNA named DlMADS8 was isolated from the young spikelets of the sweet bamboo, Dendrocalamus latiflorus by rapid amplification of cDNA end (RACE). DNA sequence analysis showed that DlMADS8 was composed of full ORF and 3'UTR, but without 5'UTR. The cDNA contained 1059 nucleotides and encoded a putative protein of 244 amino acid residues. The gene displayed the structure of a typical plant MADS-box gene, which consisted of a MADS domain, K domain, a short I region, and the C-terminal region. Phylogenetic analysis of plant MADS-box genes based on amino acid sequences revealed that DlMADS8 was grouped into the AGAMOUS-LIKE 2 (AGL2)-like subfamily. It was homologous to the LEAFY HULL STERILE1 (LHS1) genes of grasses. To study the functions of it, DlMADS8 cDNA clone driven by the CaMV 35S promoter was transformed into Arabidopsis thaliana. Transgenic plants of DlMADS8 exhibited the phenotypes of curled leaves and early flowering. After bolting, three novel phenotypes related to inflorescence development were observed in different transgenic plants. No obvious homeotic conversions of floral organs were observed in all of the 35S::DllMADS8 transgenic Arabidopsis plants. These results indicated that DlMADS8 probably plays a role in floral meristem determinacy and is involved in controlling the flowering time of D. latiflorus.     

Cloning, soluble expression, rapid purification and characterization of human Cofilin1

Cofilin1 is an actin-binding protein that plays a critical role in the regulation of actin cytoskeleton and consequently affects various physiological processes. In this study, the human Cofilin1 cDNA was cloned into the expression vector pET-28a(+) with a 6 × His tag and expressed as soluble protein in Escherichia coli BL21(DE3). Approximately 78 mg of Cofilin1, which showed high activity as determined by native PAGE, could be purified from each liter of LB medium by His-tag affinity chromatography and gel filtration. Further, high-titer IgG against Cofilin1 was positively detected after immunization in rabbits and the polyclonal antibodies were purified and identified. Together, this report provides the first protocol to efficiently obtain human Cofilin1 with high biological activity and immunogenicity using E. coli BL21 (DE3) expression system.     

Cloning,expression and characterization of a novel human VMP gene*

We report here cloning and characterization of a novel human gene, termed VMP, which is a vesicular membrane protein. RT-PCR analysis shows that VMP is expressed exclusively in brain of the 16 tissues examined, suggesting that it is a neuron-specific membrane protein. The cDNA encodes 195 amino acid with a putative molecular weight of about 24 KDa. VMP contains two putative membrane spanning domains and a hydrophilic tail homologous to the microtubule-binding domain of MAPs. So it is speculated that VMP may associated with microtubules through its C-terminal and plays an important role in vesicular organelles transport and nerve signals.     

Expression of a novel human sialidase encoded by the NEU2 gene

Sialidases (E.C.3.2.1.18) belong to a group of glycohydrolytic enzymes, widely distributed in nature, which remove sialic acid residues from glycoproteins and glycolipids. All of the sialidase so far characterized at the molecular level share an Asp block, repeated three to five times in the primary structure, and an F/YRIP sequence motif which is part of the active site. Using a sequence homology-based approach, we previously identified a human gene, named NEU2, mapping to chromosome 2q37. NEU2 encoded protein is a polypeptide of 380 amino acids with two Asp block consensuses and the YRIP sequence in the amino terminal part of the primary structure. Here we demonstrate that NEU2 encodes a functional sialidase. NEU2 was expressed in COS7 cells, giving rise to a dramatic increase in the sialidase activity measured in cell extracts with the artificial substrate 4-MU-NANA. Using a rabbit polyclonal antiserum, on Western blots a protein band with a molecular weight of about 42 kDa was detectable, and its cytosolic localization was demonstrated with cell fractionation experiments. These results were confirmed using immunohistochemical techniques. NEU2 expression in E.coli cells allowed purification of the recombinant protein. As already observed in the enzyme expressed in COS7 cells, NEU2 pH optimum corresponds to 5.6 and the polypeptide showed a K(m)for 4-MU-NANA of 0.07 mM. In addition, based on the detectable similarities between the NEU2 amino acid sequence and bacterial sialidases, a prediction of the three-dimensional structure of the enzyme was carried out using a protein homology modeling approach.     

Cloning and expression of a soluble sialidase from Chinese hamster ovary cells: sequence alignment similarities to bacterial sialidases

A cDNA encoding a soluble sialidase from Chinese hamster ovary(CHO) cells has been cloned and expressed. Completely degenerateoligonucleotide primers, which were based on the amino acidsequence of peptides obtained from the purified sialidase (Warneret al., Glycobiology, 3, 455–463, 1993), and the polymerasechain reaction, with single-stranded cDNA template, were employedto generate a unique oligonucleotide probe. The unique probeof 93 bp was used for screening a     

人RHD基因的克隆和鉴定

目的克隆人RHD基因,并对其进行鉴定。方法以RhD阳性志愿者骨髓为材料,用TRIzol试剂提取总RNA;设计、合成人RHD基因扩增引物,RT-PCR方法扩增RHD基因片段;T／A克隆后将其亚克隆人pET28a（＋）载体中,经酶切、PCR和测序对重组质粒进行鉴定。结果骨髓总RNA被成功提取;RT-PCR成功扩增出RHD基因片段,其大小与预期约509bp基本一致;T／A克隆后再将其亚克隆,通过酶切和PCR证明RHD基因成功亚克隆入pET28a（＋）载体中;基因测序结果比对显示,与已公布的RHD基因（GenBank登录号为NM016124）序列基本一致,同源性为98％。结论成功克隆了RHD基因,这将为进一步研究奠定基础。     

Cloning, characterization, and heat stress-induced redistribution of a protein homologous to human hsp27 in the zebrafish Danio rerio

Hsp27 is a small heat shock protein (shsp) regulating stress tolerance and increasingly thought to play roles in tissue homeostasis and differentiation. The zebrafish Danio rerio is an important model for the study of developmental processes, but little is known regarding shsps in this animal. Here, we report the sequence, expression, regulation, and function of a zebrafish protein (zfHsp27) homologous to human Hsp27. zfHsp27 contains three conserved phosphorylatable serines and a cysteine important for regulation of apoptosis, but it lacks much of a C-terminal tail domain and shows low homology in two putative actin interacting domains that are features of mammalian Hsp27. zfHsp27 mRNA is most abundant in adult skeletal muscle and heart and is upregulated during early embryogenesis. zfHsp27 expressed in mammalian fibroblasts was phosphorylated in response to heat stress and anisomycin, and this phosphorylation was prevented by treatment with SB202190, an inhibitor of p38 MAPK. Expression of zfHsp27 and human Hsp27 in mammalian fibroblasts promoted a similar degree of tolerance to heat stress. zfHsp27 fusion proteins entered the nucleus and associated with the cytoskeleton of heat stressed cells in vitro and in zebrafish embryos. These results reveal conservation in regulation and function of mammalian and teleost Hsp27 proteins and define zebrafish as a new model for the study of Hsp27 function.     

Cloning and characterization of a novel human TEKTIN1 gene

Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. A new member of the tektin gene family was cloned from the human fetal brain cDNA library. We hence named it the human TEKTIN1 gene. TEKTIN1 cDNA consists of 1375 bp and has a putative open reading frame encoding 418 amino acids. The predicted protein is 48.3 kDa in size, and its amino acid sequence is 82% identical to that of the mouse, rat, and dog. One conserved peptide RPNVELCRD was observed at position number 323–331 of the amino acid sequence, which is a prominent feature of tektins and is likely to represent a functionally important protein domain. TEKTIN1 gene was mapped to the human chromosome 17 by BLAST search, and at least eight exons were found. Northern blot analysis indicated that TEKTIN1 was predominantly expressed in testis. By in-situ hybridization analysis, TEKTIN1 mRNA was localized to spermatocytes and round spermatids in the seminiferous tubules of the mouse testis, indicating that it may play a role in spermatogenesis.