Paragonimus westermani: molecular cloning,expression, and characterization of a recombinant yolk ferritin

Ferritin is an intracellular protein involved in iron metabolism. A cDNA PwYF-1 cloned from the adult Paragonimus westermani cDNA library encoded a putative polypeptide of 216 amino acids homologous with ferritins of vertebrates and invertebrates. Febinding motifs identified in PwYF-1 polypeptide were conserved and predicted to form a ferroxidase center. PwYF-1 polypeptide contained an extended peptide of 45 amino acids at its C-terminus. Recombinant PwYF-1 protein, expressed and purified from Escherichia coli, showed iron-uptake ability and ferroxidase activity. Ferroxidase activity of recombinant PwYF-1 protein was reactivated by secondary addition of apotransferrin to assay mixture. Mouse immune serum raised against the recombinant PwYF-1 protein recognized specifically 24 kDa protein from adult P. westermani lysate. PwYF-1 protein was localized to vitelline follicles and the eggs of P. westermani. Collectively, PwYF-1 protein was identified as a P. westermani yolk ferritin.     

巴西橡胶树水通道蛋白基因cDNA的克隆及序列分析

采用简并性RT-PCR和RACE技术,从巴西橡胶树(Hevea brasiliensis)中克隆了2个aquaporin基因的全长cDNA,分别命名为HbPIP1和HbPIP2(GenBank登录号分别为GQ479823和GQ479824).测序和生物信息学分析表明,HbPIP1和HbPIP2分别编码287和278个氨基酸,均具有6个跨膜区,包含MIP家族信号序列SGX-HXNPAVT/SFG、高等植物PIP高度保守序列GGGANXXXXGY和TGI/TNPARSL/FGAAI/VI/VF/YN.核酸和氨基酸序列的同源性分析表明,HbPIP1和HbPIP2均属于水通道蛋白基因家族的新成员.     

两个菜花烙铁头蛇毒金属蛋白酶去整合素基因的克隆与序列分析(英文)

从菜花烙铁头蛇的毒腺中 ,利用RT PCR进行体外扩增 ,克隆到 2个金属蛋白酶 去整合素基因 ,命名为TJM 1、TJM 2 .TJM 1cDNA全长为 15 2 8bp ,编码 4 81个氨基酸 ;TJM 2cDNA全长为 15 78bp ,编码 4 84个氨基酸 .TJM 1和TJM 2都属于Ⅱ型蛇毒金属蛋白酶 ,由信号肽、前肽、金属蛋白酶、间隔肽和去整合素 5部分组成 .Ⅱ型蛇毒金属蛋白酶氨基酸序列的比较及进化分析显示 ,它可进一步分为两类 ,一类包括大多数Ⅱ型蛇毒金属蛋白酶 (其中含有TJM 1) ,而TJM 2和agkistin则组成了另一类 .并且TJM 2和agkistin的第 4 0 7位和第 4 2 6位残基都是半胱氨酸 ,而在其它Ⅱ型金属蛋白酶的相应位置 ,4 0 7位是丝氨酸 ,4 2 6位则缺失 .TJM 2和agkistin均有可与整合素α2 Ⅰ区域特异性结合的片段Q NRKRHDNAQ(残基 2 76～ 2 84 ) ,这个片段在其它Ⅱ型金属蛋白酶中并没有发现 .因此推断 ,TJM 2和agkistin可能属于一类新型的Ⅱ型蛇毒金属蛋白酶 .     

Isolation and analysis of two cellulase cDNAs from Orpinomyces joyonii

Two cellulase cDNAs, celB29 and celB2, were isolated from a cDNA library derived from mRNA extracted from the anaerobic fungus, Orpinomyces joyonii strain SG4. The nucleotide sequences of celB2 and celB29 and the primary structures of the proteins encoded by these cDNAs were determined. The larger celB29 cDNA was 1966bp long and encoded a 477 amino acid polypeptide with a molecular weight of 54kDa. Analysis of the 1451bp celB2 cDNA revealed an 1164bp open reading frame coding for a 44kDa protein consisting of 388 amino acids. Both deduced proteins had a high sequence similarity in central regions containing putative catalytic domains. Primary structure analysis revealed that CelB29 contained a Thr/Pro-rich sequence that separated the N-terminal catalytic domain from a C-terminal reiterated region of unknown function. Homology analysis showed that both enzymes belong to glycosyl hydrolase family 5 and were most closely related to endoglucanases from the anaerobic fungi Neocallimastic patriciarum, Neocallimastix frontalis and Orpinomyces sp. The classification of CelB29 and CelB2 as endoglucanases was supported by enzyme assays. The cloned enzymes had high activities towards barley beta-glucan, lichenan and carboxymethylcellulose (CMC), but not Avicel, laminarin, pachyman, xylan and pullulan. In addition, CelB29 and CelB2 showed activity against p-nitrophenyl-beta-D-cellobioside (pNP-G(2)) to p-nitrophenyl-beta-D-cellopentaoside (pNP-G(5)) but not p-nitrophenyl-beta-D-glucopyranoside (pNP-G(1)) with preferential activity against p-nitrophenyl-beta-D-cellotrioside (pNP-G(3)). Based on these results, we proposed that CelB29 and CelB2 are endoglucanases with broad substrate specificities for short- and long-chain beta-1,4-glucans.     

Cloning of a lectin cDNA and seasonal changes in levels of the lectin and its mRNA in the inner bark ofRobinia pseudoacacia

A cDNA clone encoding a lectin was isolated by immunological screening of an expression library prepared from poly(A)⁺ RNA from the inner bark ofRobinia pseudoacacia. The cDNA clone (RBL104) had an open reading frame of 858 bp that encoded a polypeptide with a predicted molecular weight of 31210. This molecular weight corresponded closely to that of a polypeptide immunoprecipitated from products of translationin vitro of the poly(A)⁺ RNA. Thus, RBL104 appeared to be a full-length cDNA. The N-terminal amino acid sequence of the purified lectin protein matched a portion of the predicted amino acid sequence. It appeared that the lectin was synthesized as a precursor that consisted of a putative signal peptide of 31 amino acids and a mature polypeptide of 255 amino acids. Southern blot analysis of the genomic DNA revealed that the lectin was encoded by a small multigene family. The lectin was mostly localized in the axial and ray parenchymal cells of the inner bark. A small amount of lectin was also found in the axial and ray parenchymal cells of the xylem. The lectin accumulated in the inner bark in September, remained at high levels during the winter and disappeared in May. The mRNA for the lectin was detected from August to the following March. The appearance and disappearance of the mRNA were observed prior to those of the lectin protein.     

Proteus mirabilis amino acid deaminase: cloning, nucleotide sequence, and characterization of aad.

Proteus, Providencia, and Morganella species produce deaminases that generate alpha-keto acids from amino acids. The alpha-keto acid products are detected by the formation of colored iron complexes, raising the possibility that the enzyme functions to secure iron for these species, which do not produce traditional siderophores. A gene encoding an amino acid deaminase of uropathogenic Proteus mirabilis was identified by screening a genomic library hosted in Escherichia coli DH5 alpha for amino acid deaminase activity. The deaminase gene, localized on a cosmid clone by subcloning and Tn5::751 mutagenesis, was subjected to nucleotide sequencing. A single open reading frame, designated aad (amino acid deaminase), which appears to be both necessary and sufficient for deaminase activity, predicts a 473-amino-acid polypeptide (51,151 Da) encoded within an area mapped by transposon mutagenesis. The predicted amino acid sequence of Aad did not share significant amino acid sequence similarity with any other polypeptide in the PIR or SwissProt database. Amino acid deaminase activity in both P. mirabilis and E. coli transformed with aad-encoding plasmids was not affected by medium iron concentration or expression of genes in multicopy in fur, cya, or crp E. coli backgrounds. Enzyme expression was negatively affected by growth with glucose or glycerol as the sole carbon source but was not consistent with catabolite repression.     

Cloning, sequencing and expression of the glutamine synthetase structural gene (glnA) from the obligate methanotroph Methylococcus capsulatus (Bath)

The structural gene (glnA) encoding the ammonia-assimulation enzyme glutamine synthetase (GS) has been cloned from the obligate methanotroph Methylococcus capsulatus (Bath). Complementation of Escherichia coli glnA mutants was demonstrated. In vitro expression analysis revealed that the cloned glnA gene coded for a polypeptide of apparent Mr 60,000, as determined by PAGE. Expression of the M. capsulatus (Bath) glnA gene in E. coli was regulated by nitrogen levels in an Ntr+ but not an Ntr- background. The nucleotide sequence of the M. capsulatus (Bath) glnA gene and flanking sequences was determined. This gene, of 1407 bp, encoded a polypeptide of Mr 51717 containing 468 amino acids. The 5' leader region contained three putative promoters. Promoters P1 and P3 resembled the canonical -10 -35 E. coli-type promoter. Promoter P2, which was located between P1 and P3, resembled the NtrA-dependent promoters of enteric organisms. A potential NtrC-binding site was also determined, flanking the Pribnow box at P1. Comparisons of nucleotide-derived amino acid sequences of GS enzymes from prokaryotes and eukaryotes with GS from M. capsulatus are made.     

The antigenic and catalytically active formamidase of Paracoccidioides brasiliensis: protein characterization, cDNA and gene cloning, heterologous expression and functional analysis of the recombinant protein

Paracoccidioides brasiliensis is a well-characterized pathogen of humans. To identify proteins involved in the fungus-host interaction, P. brasiliensis yeast proteins were separated by liquid isoelectric focusing, and fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Immunoreactive bands were detected with pooled sera of patients with P. brasiliensis infection. A protein species with a molecular mass of 45 kDa was subsequently purified to homogeneity by preparative gel electrophoresis. The amino acid sequence of four endoproteinase Lys-C-digested peptides indicated that the protein was a formamidase (FMD) (E.C. 3.5.1.49) of P. brasiliensis. The complete cDNA and a genomic clone (Pbfmd) encoding the isolated FMD were isolated. An open reading frame predicted a 415-amino acid protein. The sequence contained each of the peptide sequences obtained from amino acid sequencing. The Pbfmd gene contained five exons interrupted by four introns. Northern and Southern blot analysis suggested that there is one copy of the gene in P. brasiliensis and that it is preferentially expressed in mycelium. The complete coding cDNA was expressed in Escherichia coli to produce a recombinant fusion protein with glutathione S-transferase (GST). The purified recombinant protein was recognized by sera of patients with proven paracoccidioidomycosis and not by sera of healthy individuals. The recombinant 45-kDa protein was shown to be catalytically active; FMD activity was detected in P. brasiliensis yeast and mycelium.     

Cloning and expression of a plant homologue of the small subunit of the Golgi-associated clathrin assembly protein AP19 from Camptotheca acuminata

Clathrin-coated vesicles (CCVs) are involved in selective protein transport in eukaryotes. AP-1 and AP-2 are protein complexes found in the CCVs of the Golgi apparatus and the plasma membrane respectively. AP19 is the smallest polypeptide chain components of AP-1. We have identified a cDNA clone (CAP19) encoding a putative homologue for the assembly protein AP19 from the Chinese medicinal tree, Camptotheca acuminata. The deduced polypeptide contains 161 amino acids and has a predicted M _r of 18 820. DNA blot analysis suggests that the AP19s of C. acuminata are encoded by a small gene family. CAP19 was expressed ubiquitously throughout the plant suggesting that it may be involved in general Golgi-mediated secretion.     

Vaccinia virus encodes a thymidylate kinase gene: sequence and transcriptional mapping

The nucleotide sequence and deduced amino acid sequence of a vaccinia virus gene from the SalI F fragment are shown. The predicted polypeptide shares 42% amino acid identity over a 200 amino acid region with Saccharomyces cerevisiae thymidylate kinase (TmpK) and has low homology with herpes simplex virus deoxypyrimidine kinase. Northern blotting and S1 nuclease protection showed that the TmpK gene is transcribed early during infection and mapped the mRNA 5' end to immediately upstream of the second inframe ATG codon of the open reading frame (ORF). The encoded polypeptide is predicted to be 204 amino acids long (23.2 kD) and is almost colinear with yeast TmpK. Vaccinia virus possesses genes for TK and TmpK, separated by 57 kilobases of DNA, which are co-ordinately expressed and the encoded enzymes perform sequential steps in the same biochemical pathway.     

Mouse cathepsin F: cDNA cloning, genomic organization and chromosomal assignment of the gene

A murine cysteine protease of the papain family was identified by dbEST-database search. A 1.87kb full-length cDNA encoding a predicted polypeptide of 462 amino acids was sequenced. Since the encoded polypeptide shows more than 80% sequence identity with human cathepsin F, it is most likely that this cDNA represents the murine homologue of cathepsin F, and it was therefore named accordingly. Murine cathepsin F exhibits a domain structure typical for papain-like cysteine proteases, a 20 amino acid N-terminal hydrophobic signal sequence followed by an extraordinarily long propeptide of 228 amino acids and the domain of the mature protease comprising 214 amino acids. The mature region contains all features characteristic of a papain-like cysteine protease, including the highly conserved cysteine, histidine and asparagine residues of the 'catalytic triad'. Genomic clones covering the murine cathepsin F gene were isolated. The mouse cathepsin F gene consists of 14 exons and 13 introns and spans 5.8kb. Murine cathepsin F was mapped to chromosome 19, a region with synteny homology to a region of human chromosome 11 to which human cathepsin F has been mapped previously. Northern blot analysis of RNA from multiple tissues revealed a ubiquitous expression of cathepsin F in mouse and man.     

N-terminal and cDNA characterization of murine lymphocyte antigen Ly-6C.2

The Ly-6C.2 molecule was purified from K36 tumor cells by affinity chromatography and gel filtration. The electrophoretically homogeneous preparation, with m.w. 15,000, was tested with a panel of antibodies that confirmed the presence of the LY-6C.2 epitope. An N-terminal sequence of 39 amino acids was obtained showing 59% homology with the corresponding portion of the Ly-6A.2 polypeptide. Based on the least homologous (29%) 14 amino acid segment, an oligonucleotide probe was constructed, and Ly-6C.2 cDNA was cloned from a BW5147 cDNA library. A 794-base pair cDNA containing the entire coding region had 82% homology with Ly-6A.2 cDNA. The encoded polypeptide sequence of 131 amino acids containing a perfect correlation with the N-terminal sequence data was 63% homologous with that of Ly-6A.2. The greatest homology was in the leader, first 16 N-terminal and last 39 C-terminal amino acids. The latter are likely to be important in determining the attachment of glycophosphatidylinositol. Despite results indicating fewer disulfide constraints in the Ly-6C molecule, the predicted sequence contains 10 cysteine residues nearly perfectly matched with those predicted in Ly-6A.     

The brain link protein-1 (BRAL1): cDNA cloning, genomic structure, and characterization as a novel link protein expressed in adult brain

We report here molecular cloning and expression analysis of the gene for a novel human brain link protein-1 (BRAL1) which is predominantly expressed in brain. The predicted open reading frame of human brain link protein-1 encoded a polypeptide of 340 amino acids containing three protein modules, the immunoglobulin-like fold and proteoglycan tandem repeat 1 and 2 domains, with an estimated mass of 38 kDa. The brain link protein-1 mRNA was exclusively present in brain. When analyzed during mouse development, it was detected solely in the adult brain. Concomitant expression pattern of mRNAs for brain link protein-1 and various lectican proteoglycans in brain suggests a possibility that brain link protein-1 functions to stabilize the binding between hyaluronan and brevican. The human BRAL1 gene contained 7 exons and spanned approximately 6 kb. The entire immunoglobulin-like fold was encoded by a single exon and the proteoglycan tandem repeat 1 and 2 domains were encoded by a single and two exons, respectively. The deduced amino acid sequence of human brain link protein-1 exhibited 45% identity with human cartilage link protein-1 (CRTL1), previously reported as link protein to stabilize aggregates of aggrecan and hyaluronan in cartilage. These results suggest that brain link protein-1 may have distinct function from cartilage link protein-1 and play specific roles, especially in the adult brain.     

Molecular cloning and sequencing of cDNAs encoding the proteolipid subunit of the vacuolar H(+)-ATPase from a higher plant.

To understand the molecular structure of the vacuolar H(+)-translocating ATPase from plants, cDNAs encoding the N,N'-dicyclohexylcarbodiimide-binding 16-kDa proteolipid from oat (Avena sativa L. var. Lang) have been obtained. A synthetic oligonucleotide corresponding to a region of the bovine proteolipid cDNA (Mandel, M., Moriyama, Y., Hulmes, J.D., Pan, Y.-C.E., Nelson, H., and Nelson, N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5521-5524) was used to screen an oat cDNA library constructed in lambda gt11. The nucleotide sequences of several positive clones (VATP-P1, clones 12, 54, 93) demonstrated the presence of a small multigene family. The four clones showed extensive divergence in their codon usage and their 3'-untranslated regions; however, the deduced amino acid sequences of the proteins were 97-99% identical. These clones encoded the proteolipid subunit as one of them (clone 12) expressed a fusion protein that reacted with an antibody to the 16-kDa proteolipid. The open reading frame of one cDNA clone (VATP-P1) predicted a polypeptide of 165 amino acids with a molecular mass of 16,641. Based on hydropathy plots, a molecule with four membrane-spanning domains was predicted, in which domain IV was especially conserved among different species. This domain showed 80% identity in nucleotide or amino acid sequences between the oat and the bovine proteolipids and contained a glutamate residue that is the putative N,N'-dicyclohexylcarbodiimide-binding residue. The presence of a small multigene family of the 16-kDa proteolipid was confirmed by Southern blot analysis showing that several distinct restriction fragments of oat nuclear DNA hybridized with the VATP-P1 cDNA.