Cloning of a family of serine protease genes from the cat flea Ctenocephalides felis

Serine protease gene fragments approximately 480 nucleotides in length were amplified from Ctenocephalides felis larval and adult cDNA libraries using degenerate oligonucleotide PCR primers. Partial clones of thirty-eight distinct serine protease encoding sequences were isolated, and nineteen different full-length cDNAs encoding mature serine proteases were subsequently cloned and sequenced. All of the mature proteases contained the histidine, aspartic acid and serine amino acids of the catalytic triad characteristic of serine proteases. The mature C. felis serine proteases had amino acid sequences that were at most 29–53% identical to those known insect and arachnid serine proteases. Two of the C. felis gene sequences had similarity with the Drosophila melanogaster developmental genes snake and stubble. mRNA expression of selected serine protease genes was examined in different life stages, tissues, genders, and in response to bloodfeeding.     

The white gene from the yellow fever mosquito, Aedes aegypti

We report the cloning and primary characterization of both cDNA and genomic fragments from the white gene of the yellow fever mosquito, Aedes aegypti . Comparisons of the conceptual translation product with white genes from four other species within the order Diptera show that the Ae. aegypti gene is most similar to the white gene of the mosquito vector of human malaria, Anopheles gambiae (86% identity and 92% similarity). The analysis of the primary sequence of genomic DNA at the 5'-end of the coding region revealed the presence of an intron that is also present in An. gambiae , but not in the vinegar fly, Drosophila melanogaster . The isolated clones of the Ae. aegypti white gene will enable the construction of a marker gene for use in the development of a germline transformation system for this species.     

胎儿间充质干细胞cDNA扣除文库的构建

为了获得胎儿和成人间充质干细胞（mesenchymal stem cells,MSC)的差异表达基因，尤其是在胎儿MSC中特异表达的基因，应用抑制扣除杂交技术构建了胎儿和成人MSC cDNA扣除库。首先，分别从胎儿和成人MSC中提取总RNA，按照SMART PCR方法依次合成单链和双链cDNA，经Rsa I酶切后，胎儿MSC cDNA分别与两种不同的接头连接，再与成人MSC cDNA经过两次扣除杂交及两轮抑制性PCR扩增后，将产物与T载体连接构建成cDNA扣除库，并转染大肠杆菌进行库扩增。结果：库扩增后共取得890个克隆，对所获得的克隆进行PCR扩增鉴定，获得768个阳性克隆，阳性率为86．3％。插入片段大小分布在0.2-1kb，平均为400－600bp。结论：抑制扣除杂交技术是一种简便、有效的筛选差异表达基因的方法。胎儿MSC cDNA扣除库的构建为进一步筛选、克隆胎儿MSC中特异性表达的功能相关基因奠定了基础。     

Isolation and characterization of insect PC2-like prohormone convertase cDNA

Endoproteolytic processing of large precursor molecules at basic amino acid residues plays an important role in the maturation of many hormones, neuropeptides and other regulatory proteins. Enzymes performing these reactions are designated as prohormone or proprotein convertases and belong to the subtilisin family of serine proteases. The screening of a larval cDNA library of the sheep blowfly Lucilia cuprina resulted in the isolation of two cDNAs encoding a PC2-like prohormone convertase. The predicted 675 amino acid preproprotein (LcuPC2) exhibits its highest identity to invertebrate and vertebrate prohormone convertase 2 homologues, and a noticeably lower identity to the so far known insect furin-like prohormone convertases of Drosophila melanogaster and Aedes aegypti. In Northern blot experiments a signal at 2.5 kb could be detected.     

Isolation and sequence analysis of serine protease cDNAs from mouse cytolytic T lymphocytes

Three new cDNA clones (designated MCSP-1, MCSP-2, and MCSP-3) encoding mouse serine proteases were isolated from cloned cytolytic T lymphocytes (CTL) by a modified differential screening procedure. The putative mature proteins of MCSP-2 and MCSP-3 are each composed of 228 amino acids with molecular weights of 25,477 and 25,360, respectively. NH2-terminal amino acids of MCSP-2- and MCSP-3-predicted proteins were identical to those reported for granzyme E and F, respectively. The third species, MCSP-1, was closely related to the two other cDNA species but approximately 30 amino acids equivalents of the NH2- terminal portion of the cDNA were not cloned. The amino acids forming the active sites of serine proteases were well conserved among the three predicted proteins. The active site pocket residue positioned six residues before the active-site Ser184 is alanine in MCSP-1, threonine in MCSP-2, and serine in MCSP-3, indicating that both MCSP-2 and MCSP-3 may have chymotrypsin-like specificity. There are three potential asparagine-linked glycosylation sites in MCSP-1 and MCSP-3, and four in MCSP-2-deduced amino acid sequences. Amino acid comparison of MCSP-1 with four other reported serine proteases whose active site pocket residue is alanine revealed that MCSP-1 was substantially different from the other molecules, indicating that MCSP-1 may be a new member of mouse T cell serine protease family. Antibodies made against a MCSP-1 lacZ gene fusion protein stain granules of CTL and react on immunoblots with two distinct granule protein bands of 29 and 35-40 kD. Only the 35- kD species labels with [3H]DFP. Since a protease cascade may play a key role in cytolytic lymphocyte activation, our isolation of cDNAs representative of unique serine esterases should help to investigate such a cascade process.     

Structure of the human B lymphocyte receptor for C3d and the Epstein- Barr virus and relatedness to other members of the family of C3/C4 binding proteins [published erratum appears in J Exp Med 1988 Nov 1;168(5):1953-4]

Human complement receptor type 2 (CR2) is the B lymphocyte receptor for C3d and the Epstein-Barr virus. This protein is also a member of a family of C3b/C4b binding proteins that regulate complement activation, comprise tandemly repeated 60-75 amino acid sequences, and whose genes map to band q32 on chromosome 1. Overlapping cDNA clones encoding the entire human CR2 protein have been isolated from a human tonsillar cDNA library. The derived amino acid sequence of 1,032 residues encodes a peptide of 112,716 mol wt. A signal peptide was identified, followed by 15 copies of the short consensus repeat (SCR) structure common to the C3/C4 binding protein family. The entire extracellular portion of the protein comprised SCRs, thus, the ligand binding sites both for C3d and the EBV protein gp350/220 are positioned within this structure. Immediately following the final SCR was a transmembrane sequence of 24 amino acids and a cytoplasmic region of 34 amino acids. One of five cDNA clones isolated contained an additional SCR, providing evidence for alternative mRNA splicing or gene products of different human alleles. The CR2 cDNAs were used to isolate CR2-specific genomic phage. The entire CR2 coding sequences were found within 20 kb of human DNA. Analysis of the CR2 cDNA sequence indicated that CR2 contained internally homologous regions and suggested that CR2 arose by duplication of a primordial gene sequence encoding four SCRs. Comparison of the CR2 peptide sequence with those of other members of the gene family has identified many regions highly homologous with human CR1, fewer with C4bp and decay accelerating factor, and very few with factor H, and suggested that CR2 and CR1 arose by duplication of the same ancestral gene sequence. The homology between CR2 and CR1 extended to the transmembrane and cytoplasmic regions, suggesting that these sequences were derived from a common membrane-bound precursor.     

Serine proteases identified from a Costelytra zealandica (White) (Coleoptera: Scarabaeidae) midgut EST library and their expression through insect development

Costelytra zealandica larvae are pests of New Zealand pastures causing damage by feeding on the roots of grasses and clovers. The major larval protein digestive enzymes are serine proteases (SPs), which are targets for disruption in pest control. An expressed sequence tag (EST) library from healthy, third instar larval midgut tissue was constructed and analysed to determine the composition and regulation of proteases in the C. zealandica larval midgut. Gene mining identified three trypsin-like and 11 chymotrypsin-like SPs spread among four major subgroups. Representative SPs were examined by quantitative PCR and enzyme activity assayed across developmental stages. The serine protease genes examined were expressed throughout feeding stages and downregulated in nonfeeding stages. The study will improve targeting of protease inhibitors and bacterial disruptors of SP synthesis.     

丙型肝炎病毒1b亚型诊断芯片的制备与实验室研究

目的建立一种简单有效的制备、筛选丙型肝炎病毒(HCV)1b亚型cDNA基因芯片探针的技术。方法应用cDNA文库法制备芯片探针，限制性内切酶Sau3AI消化HCV-1b全长cDNA。所得的酶切片段在72℃补平加单个碱基A，然后与pMD18-T载体连接，AT克隆，用载体引物进行PCR初步鉴定，并测序。将筛选出的片段打印在氨基修饰的玻片上制备成cDNA芯片并进行杂交验证分析。样品标记采用限制性显示PCR(Restriction Display PCR RD—PCR)技术。结果应用cDNA文库法，共得到22大小相对一致(250—750bp)的基因片段，序列分析表明，均属于HCV—1b基因，可以作为诊断芯片探针；芯片杂交结果显示，样品和诊断基因芯片杂交的敏感性和特异性均佳。结论用cDNA文库法收集片段是一种快速、简便制备芯片探针的实用方法；制备的诊断芯片可以用于检测HCV-1b RNA，具有敏感、检测结果较为可靠的优点。     

cDNA cloning of human plasminogen activator-inhibitor from endothelial cells.

Full-length cDNA for plasminogen activator inhibitor (PAI-1) was isolated from a human umbilical vein endothelial cell (HUVEC) lambda gt11 cDNA library. Three overlapping clones were identified by immunologic screening of 10(6) recombinant phage using a rabbit anti-human fibrosarcoma PAI-1 antiserum. The fusion proteins encoded by these three clones also react strongly with a monoclonal mouse anti-human fibrosarcoma PAI-1 antibody. By nucleotide sequence analysis, PAI-1 cDNA encodes a protein containing 402 amino acids with a predicted, nonglycosylated molecular mass of 45 kD. Identity of this material as authentic PAI-1 was confirmed by the presence of high level homology with the primary amino acid sequence of an internal peptide prepared from purified rat hepatoma PAI-1. The predicted amino acid sequence also reveals extensive homology with other members of the serine protease inhibitor gene family. Cultured HUVECs contain two PAI-1 mRNA species, both encoded by a single gene, differing by 1 kb in the 3' untranslated region. The PAI-1 gene is located on human chromosome 7.     

Secretory Granule Proteases in Rat Mast Cells. Cloning of 10 Different Serine Proteases and a Carboxypeptidase A from Various Rat Mast Cell Populations

Two of the major rat mast cell proteases, rat mast cell protease 1 (RMCP-1) and RMCP-2, have for many years served as important phenotypic markers for studies of various aspects of mast cell (MC) biology. However, except for these proteases only fragmentary information has been available on the structure and complexity of proteases expressed by different subpopulations of rat MCs. To address these questions, cDNA libraries were constructed from freshly isolated rat peritoneal MCs and from the rat mucosal MC line RBL-1. cDNA clones for 10 different serine proteases (RMCP-1-10), and the MC carboxypeptidase A were isolated and characterized. Six of these proteases have not been isolated previously. Based on their protease content, three separate subpopulations of MCs were identified. Connective tissue MCs (CTMCs) from the ear and peritoneum express the chymases RMCP-1 and -5, the tryptases RMCP-6, and -7 and the carboxypeptidase A. However, based on a large difference in the level of expression of RMCP-7, CTMCs of these two organs may be regarded as two separate subpopulations. RMCP-2 and the three closely related proteases of the RMCP-8 subfamily were identified as the major mucosal MC proteases in rat. In contrast to what has been reported for human MCs, no expression of cathepsin G or cathepsin G–like proteases was detected in any of the rat MC populations. To determine mRNA frequencies for the various proteases expressed by normal tissue MCs, an unamplified peritoneal MC cDNA library was screened with a panel of monospecific cDNA probes. These results showed that peritoneal MCs are highly specialized effector cells with mRNA frequencies for the major proteases in the range of several percent of the total mRNA pool.     

A high proportion of T lymphocytes that infiltrate H-2-incompatible heart allografts in vivo express genes encoding cytotoxic cell-specific serine proteases, but do not express the MEL-14-defined lymph node homing receptor

The role of cytotoxic cells in in vivo immune functions such as allograft rejection is unknown. To begin to assess the function of cytolytic cells in vivo we have begun with cytolytic cell-specific functional molecules: we have isolated and characterized cytolytic cell-specific cDNA clones from cytolytic T cell clones, both encoding distinct serine esterases. The HF gene encodes a trypsin-like enzyme while the C11 gene encodes an enzyme with likely specificity for acidic residues. Here we demonstrate, using in situ hybridization with RNA probe, that both genes are expressed selectively in a subset of T lymphocytes that have infiltrated cardiac allografts. The phenotype of these cells is consistent with the most frequent phenotype of active CTL raised in vitro: they are predominantly CD4-, CD8+, MEL-14- T cell blasts. Thus the expression of these genes, each of which encodes serine esterase found in killer cell granules in vitro, is a valid marker for these cells in vivo as well. The kinetics of their accumulation is consistent with, but not proof of, a putative role in allograft rejection. It is likely that HF and C11 gene expression will be of diagnostic value.     

A salivary serine protease of the haematophagous reduviid Panstrongylus megistus: sequence characterization,expression pattern and characterization of proteolytic activity

A cDNA encoding a trypsin‐like protease from the salivary glands of the haematophagous reduviid Panstrongylus megistus was cloned and sequenced. The deduced protein sequence showed similarities to serine proteases of other hemipterans but with substitutions in the catalytic triad and the substrate binding site. The expression of the gene increased more than sixfold after feeding. Saliva showed the highest proteolytic activity at neutral to slightly basic pH. Substrate and inhibitor profiles and zymography indicated the presence of a trypsin‐like protease with preference for Arg and Lys at P1. Using chromatography, a fibrinolytic enzyme was purified whose sequence was identified by tandem mass spectrometry as that encoded by the cDNA.     

An estimate of the number of serine protease genes expressed in sheep blowfly larvae (Lucilia cuprina)

A large and diverse family of serine protease genes was identified in first-instar larval cDNA of the sheep blowfly (Lucilia cuprina). This complex repertoire of genes was identified via a PCR approach using highly degenerate primers based on structurally conserved regions which surround the active site His and Ser residues found in all serine proteases. PCR products from entire first-instar larval cDNA, or from third-instar larval salivary glands or cardia, generated using a microscale RT-PCR method, were cloned into a plas-mid vector. Comparison of the restriction fragment patterns of PCR products generated from the three different sources suggests a highly diverse tissue-specific pattern of serine protease expression in this organism. Detailed analysis of the restriction fragment patterns of sixty-nine randomly selected clones from entire first-instar larvae revealed forty-nine different classes of PCR product. Maximum likelihood analysis of these data indicate that between 125 and 220 different serine protease genes are expressed in first-instar larvae of L. cuprina. DNA sequence analysis of ten randomly-selected clones, derived from the three tissue sources, indicated that all ten encoded serine protease gene fragments. A frequently occurring PCR product, generated from both first-instar total cDNA and third-instar cardia cDNA, showed 73% amino acid identity to a digestive protease expressed in Droso-phila melanogaster larval gut cells.