Cloning and sequence analysis of cDNA encoding aspartate aminotransferase isozymes from Panicum miliaceum L., a C4 plant.

The cytosolic and mitochondrial isozymes of aspartate aminotransferase (AspAT) function in the C4 dicarboxylate cycle of photosynthesis. We constructed a cDNA library from leaf tissues of Panicum miliaceum, an NAD-malic-enzyme-type C4 plant and screened the library for AspAT isozymes. A full-length cDNA clone for cytosolic AspAT was isolated. This clone contains an open reading frame that encodes 409 amino acids. We also isolated two cDNA clones for different precursors of mitochondrial AspAT. Comparing these two sequences in the coding regions, we found 12 amino acid substitutions out of 28 base substitutions. The encoded amino acid sequences predict that mitochondrial AspAT are synthesized as precursor proteins of 428 amino acid residues, which each consist of a mature enzyme of 400 amino acid residues and a 28-amino-acid presequence. This prediction coincides with the observation that the in vitro translation product of the mRNA for mitochondrial AspAT was substantially larger than the mature form. A comparison of the amino acid sequences of the AspAT isozymes from P. miliaceum with the published sequences for the enzymes from various animals and microorganisms reveals that functionally and/or structurally important residues are almost entirely conserved in all AspAT species.     

Corn kernel cysteine proteinase inhibitor as a novel cystatin superfamily member of plant origin. Molecular cloning and expression studies.

A full-length cDNA clone for a cysteine proteinase inhibitor (cystatin) was isolated from a lambda gt10 cDNA library of immature corn kernels by screening with a mixture of cDNA inserts for oryzacystatins I and II. The cDNA clone spans 960 base pairs, encoding a 135-amino-acid protein containing a signal peptide fragment. The protein, named corn cystatin I, is considered to be a member of the cystatin superfamily, since it contains the commonly conserved Gln-Val-Val-Ala-Gly region that exists in most known cystatins as a probable binding site and is significantly similar to other cystatins in its overall amino acid sequence. Corn cystatin I expressed in Escherichia coli showed a strong papain-inhibitory activity. Northern blot analysis showed that the amount of mRNA for corn cystatin I reaches a maximum 2 weeks after flowering and then decreases gradually.     

Cloning and sequencing of the gene encoding the candidate HtrA of Rickettsia typhi

We screened a phage library of Rickettsia typhi with a polyclonal antiserum to clone genes which encode immunogenic proteins of R. typhi. Among several clones obtained, one clone codes for a 466-amino-acid protein similar to the heat-shock protein, HtrA. The deduced rickettsial HtrA contains a putative signal peptide sequence at the N-terminus, a serine protease-like domain, and two PDZ domains. The recombinant protein of rickettsial HtrA reacted with sera from patients with murine typhus and tsutsugamushi disease. We suggest that this gene and its recombinant protein would be valuable for the immunologic diagnosis of rickettsial diseases.     

Complex formation between the NS3 serine-type proteinase of the hepatitis C virus and NS4A and its importance for polyprotein maturation.

Processing of the hepatitis C virus polyprotein is mediated by host cell signalases and at least two virally encoded proteinases. Of these, the serine-type proteinase encompassing the amino-terminal one-third of NS3 is responsible for cleavage at the four sites carboxy terminal of NS3. The activity of this proteinase is modulated by NS4A, a 54-amino-acid polyprotein cleavage product essential for processing at the NS3/4A, NS4A/4B, and NS4B/5A sites and enhancing cleavage efficiency between NS5A and NS5B. Using the vaccinia virus-T7 hybrid system to express hepatitis C virus polypeptides in BHK-21 cells, we studied the role of NS4A in proteinase activation. We found that the NS3 proteinase and NS4A form a stable complex when expressed as a single polyprotein or as separate molecules. Results from deletion mapping show that the minimal NS4A domain required for proteinase activation is located in the center of NS4A between amino acids 1675 and 1686 of the polyprotein. Amino acid substitutions within this domain destabilizing the NS3-NS4A complex also impair trans cleavage at the NS4A-dependent sites. Similarly, deletion of amino-terminal NS3 sequences impairs complex formation as well as cleavage at the NS4B/5A site but not at the NS4A-independent NS5A/5B site. These results suggest that a stable NS3-NS4A interaction is important for cleavage at the NS4A-dependent sites and that amino-terminal NS3 sequences and the central NS4A domain are directly involved in complex formation.     

The N-terminal region of the murine coronavirus spike glycoprotein is associated with the extended host range of viruses from persistently infected murine cells

Although murine coronaviruses naturally infect only mice, several virus variants derived from persistently infected murine cell cultures have an extended host range. The mouse hepatitis virus (MHV) variant MHV/BHK can infect hamster, rat, cat, dog, monkey, and human cell lines but not the swine testis (ST) porcine cell line (J. H. Schickli, B. D. Zelus, D. E. Wentworth, S. G. Sawicki, and K. V. Holmes, J. Virol. 71:9499-9507, 1997). The spike (S) gene of MHV/BHK had 63 point mutations and a 21-bp insert that encoded 56 amino acid substitutions and a 7-amino-acid insert compared to the parental MHV strain A59. Recombinant viruses between MHV-A59 and MHV/BHK were selected in hamster cells. All of the recombinants retained 21 amino acid substitutions and a 7-amino-acid insert found in the N-terminal region of S of MHV/BHK, suggesting that these residues were responsible for the extended host range of MHV/BHK. Flow cytometry showed that MHV-A59 bound only to cells that expressed the murine glycoprotein receptor CEACAM1a. In contrast, MHV/BHK and a recombinant virus, k6c, with the 21 amino acid substitutions and 7-amino-acid insert in S bound to hamster (BHK) and ST cells as well as murine cells. Thus, 21 amino acid substitutions and a 7-amino-acid insert in the N-terminal region of the S glycoprotein of MHV/BHK confer the ability to bind and in some cases infect cells of nonmurine species.     

Prothymosin alpha and parathymosin: amino acid sequences deduced from the cloned rat spleen cDNAs

A rat spleen cDNA library was screened for clones carrying the cDNAs for prothymosin alpha and parathymosin. Sequence analysis of a clone carrying the entire coding region for prothymosin alpha confirmed and completed the amino acid sequence for this polypeptide and established the number of amino acid residues as 111. Rat prothymosin alpha differs from human prothymosin alpha at six positions, including four substitutions and two insertions. The nucleotide sequences of the cDNAs for the rat and human polypeptides are more than 90% identical in the open reading frames, with significant homology extending into the 5' and 3' flanking regions. From the same library, we also isolated a clone carrying 80% of the coding region for rat parathymosin. The number of amino acid residues in rat parathymosin is 101, based on the sequence deduced from the cDNA insert and earlier information on the sequence in the amino-terminal portion of this polypeptide. Despite their similarity in size and amino acid composition, rat prothymosin alpha and rat parathymosin show only limited sequence homology, primarily in the segment including residues 14 through 25, where 10 of 12 positions are identical in the two polypeptides. this is also the region of significant sequence similarity to a 12-amino-acid segment in the p17 protein of the human immunodeficiency disease associated virus (HTLV-IIIB).     

Molecular cloning and functional expression of cDNA encoding a cysteine proteinase inhibitor,cystatin, from Job's tears (Coix lacryma-jobi L. var. Ma-yuen Stapf)

A lambdaZAP II cDNA library was constructed from mRNA in immature seeds of the grass Job's tears. A cDNA clone for a cysteine proteinase inhibitor, cystatin, was isolated from the library. The cDNA clone spanned 757 base pairs and encoded 135 amino acid residues. The deduced amino acid sequence was similar to that of cystatins from the gramineous plants rice, sorghum, and corn. The central Gln-Val-Val-Ala-Gly sequence thought to be one of the binding sites of cystatins was found. A remarkable characteristic of the peptide sequence of Job's-tears cystatin was the putative signal peptide that has been found in sorghum and corn but not in rice. The cystatin cDNA was expressed in Escherichia coli as a His-tagged recombinant protein. The purified recombinant protein inhibited papain.     

Importance of the penultimate positive charge in mouse hepatitis coronavirus A59 membrane protein

The coronavirus membrane (M) protein carboxy tail interacts with the nucleocapsid during virus assembly. Previous studies demonstrated that the two terminal residues are important, and the charged residue (R227) in the penultimate position in the mouse hepatitis coronavirus (MHV) A59 M protein was suggested to participate in intermolecular interactions with negative charges in the nucleocapsid (N) protein. To determine the significance of the positive charge at position 227, we substituted the arginine with lysine (K), aspartic acid (D), glutamic acid (E), or alanine (A) and studied these by reverse genetics in the context of a MHV full-length infectious clone. Viruses with wild-type phenotype were readily recovered with the K or A substitutions. In contrast, negative-charge substitutions were not tolerated as well. In all recovered R227D viruses the negative charge was replaced with heterologous residues resulting from apparent template switching during negative-strand synthesis of subgenomic RNA 7. An additional second-site compensatory V202I substitution was present in some viruses. Recovered R227E viruses had second-site changes within the M protein carboxy tail that were partially compensatory. Significantly, most of the second site changes in the R227E mutant viruses were previously shown to compensate for the removal of negative charges in the N protein. Our results strongly indicate that a positive charge is not absolutely required. It is clear that other regions within the tail must also be involved in helping mediate interactions between the M protein and the nucleocapsid.     

Isolation and Characterization of a cDNA That Encodes a Novel Proteinase Inhibitor I from a Tobacco Genetic Tumor

We have isolated a cDNA clone, designated GTI, by screeninga tobacco genetic tumor cDNA library with a tumor-specific "subtracted"cDNA probe. The cDNA contained the entire coding sequence fora 94-amino-acid polypeptide that exhibited significant homologyto members of the proteinase inhibitor I family from tomatoand potato. The predicted protein has a pre-sequence of 22 aminoacids but lacks a pro-sequence, unlike genes for proteinaseinhibitor I isolated to date. Furthermore, the protein encodedby GTI cDNA has a novel reactive site, having glutamine as theP₁ reactive residue. These results suggest that the GTI proteinis a novel member of the proteinase inhibitor I family. ThemRNA for GTI accumulated at a high level but only transientlyafter the wounding of tobacco plants. Thus, it appears thatthe GTI protein has a function that is related to the protectionof tissues against damage due to wounding. (Received July 22, 1992; Accepted November 3, 1992)     

Computer-assisted mutagenesis of ecotin to engineer its secondary binding site for urokinase inhibition

Inhibitors of urokinase-type plasminogen activator (uPA) were selected in vitro from two ecotin phage-display libraries to study the effect on binding of amino acid substitutions at critical positions 108, 110, 112, and 113 within the 100s loop (RNKL, respectively, in wild type ecotin). The first, a focused library, was the result of a computation-assisted approach using the three-dimensional structure of the ecotin-trypsin complex to guide the modeling of amino acid substitutions predicted to increase affinity for uPA. The second, a complete library, allowed for all substitutions at the above identified positions. The consensus sequences selected from the focused, and complete libraries were RRWS and R(R/N)QL, respectively. Inhibition constant determinations showed ecotin variants containing these sequences to be similarly potent (K(i) = 1-2 nm). These substitutions were combined with previously identified substitutions in another critical region of ecotin. One of these combinations (D70R/M84R/RRQL) is the tightest (K(i) = 50 pm) ecotin variant inhibitor of uPA. The blending of combinatorial methods and computer algorithms designed to predict stronger binders has allowed us to obtain protein derivatives that exhibit greatly increased affinity for a predetermined target. This technology can be applied to select for enhanced binding interactions at protein-protein interfaces and accelerate the process of protease inhibitor development.     

Analysis of the alpha-amylase gene of Schwanniomyces occidentalis and the secretion of its gene product in transformants of different yeast genera

We have cloned and characterized the alpha-amylase gene (AMY1) of the yeast Schwanniomyces occidentalis. A cosmid gene library of S. occidentalis DNA was screened in Saccharomyces cerevisiae for alpha-amylase secretion. The positive clone contained a DNA fragment harbouring an open reading frame of 1536 nucleotides coding for a 512-amino-acid polypeptide with a calculated Mr of 56,500. The deduced amino acid sequence reveals significant similarity to the sequence of the Saccharomycopsis fibuligera and Aspergillus oryzae alpha-amylases. The AMY l gene was found to be expressed from its original promoter in S. cerevisiae, Kluyveromyces lactis and Schizo-saccharomyces pombe leading to an active secreted gene product and thus enabling the different yeast transformants to grow on starch as a sole carbon source.     

Proteus mirabilis MR/P fimbriae: molecular cloning, expression, and nucleotide sequence of the major fimbrial subunit gene.

Proteus mirabilis, a cause of serious urinary tract infection and acute pyelonephritis, produces several putative virulence determinants, among them, fimbriae. Principally, two fimbrial types are produced by this species: mannose-resistant/Proteus-like (MR/P) fimbriae and mannose-resistant/Klebsiella-like (MR/K) fimbriae. To isolate MR/P fimbrial gene sequences, a P. mirabilis cosmid library was screened by immunoblotting and by hybridization with an oligonucleotide probe based on the N-terminal amino acid sequence of the isolated fimbrial polypeptide, ADQGHGTVKFVGSIIDAPCS. One clone, pMRP101, reacted strongly with a monoclonal antibody specific for MR/P fimbriae and with the DNA probe. This clone hemagglutinated both tannic acid-treated and untreated chicken erythrocytes with or without 50 mM D-mannose and was shown to be fimbriated by transmission electron microscopy. A 525-bp open reading frame, designated mrpA, predicted a 175-amino-acid polypeptide including a 23-amino-acid hydrophobic leader peptide. The unprocessed and processed polypeptides are predicted to be 17,909 and 15,689 Da, respectively. The N-terminal amino acid sequence of the processed fimbrial subunit exactly matched amino acid residues 24 to 43 predicted by the mrpA nucleotide sequence. The MrpA polypeptide shares 57% amino acid sequence identity with SmfA, the major fimbrial subunit of Serratia marcescens mannose-resistant fimbriae.     

cDNA clone to chick corneal chondroitin/dermatan sulfate proteoglycan reveals identity to decorin.

A 1.6-kb cDNA clone was isolated by screening a library prepared from chick corneal mRNA with a cDNA clone to bovine decorin. The cDNA contained an open reading frame coding for a M(r) 39,683 protein. A 19-amino-acid match with sequence from the N-terminus of core protein from the corneal chondroitin/dermatan sulfate proteoglycan confirmed the clone as a corneal proteoglycan and the homology with human and bovine decorin confirmed its identity as decorin. Structural features of the deduced sequence include a 16-amino-acid signal peptide, a 14-amino-acid propeptide, cysteine residues at the N- and C-terminal regions, and a central leucine-rich region (comprising 63% of the protein) containing nine repeats of the sequence LXXLXLXXNXL/I. Chick decorin contains three variations of this sequence that are tandemly linked to form a unit and three units tandemly linked to form the leucine-rich region. The presence of beta bend amino acids flanking the units may serve to delineate the units as structural elements of the leucine-rich region. Sequence homology within the repeats and the spacing of the repeats suggest that this region arose by duplication. Chick decorin primarily differs from mammalian decorins in the 19-amino-acid sequence that starts the N-terminus of the core protein. Within this region, the serine that serves as a potential acceptor for the chondroitin/dermatan sulfate side chain is preceded by a glycine instead of being followed by a glycine as it is in the mammalian decorins and all other mammalian proteoglycans.     

Structural Organization of the Gene Encoding Corn Cystatin

A genomic DNA clone encoding corn cystatin, a cysteine proteinase inhibitor of corn, was isolated from a λEMBL3 phage genomic library. The genomic DNA clone spans approximately 2.2 kb and consists of three exons and two introns. The exon number and the intron breakpoints coincide with those of the genes for two types of oryzacystatin.     

Amino acid substitutions at positions 122 and 145 of hepatitis B virus surface antigen (HBsAg) determine the antigenicity and immunogenicity of HBsAg and influence in vivo HBsAg clearance

A variety of amino acid substitutions, such as K122I and G145R, have been identified around or within the a determinant of hepatitis B surface antigen (HBsAg), impair HBsAg secretion and antibody binding, and may be responsible for immune escape in patients. In this study, we examined how different substitutions at amino acid positions 122 and 145 of HBsAg influence HBsAg expression, secretion, and recognition by anti-HBs antibodies. The results showed that the hydrophobicity, the presence of the phenyl group, and the charges in the side chain of the amino acid residues at position 145 reduced HBsAg secretion and impaired reactivity with anti-HBs antibodies. Only the substitution K122I at position 122 affected HBsAg secretion and recognition by anti-HBs antibodies. Genetic immunization in mice demonstrated that the priming of anti-HBs antibody response was strongly impaired by the substitutions K122I, G145R, and others, like G145I, G145W, and G145E. Mice preimmunized with wild-type HBsAg (wtHBsAg) or variant HBsAg (vtHBsAg) were challenged by hydrodynamic injection (HI) with a replication-competent hepatitis B virus (HBV) clone. HBsAg persisted in peripheral blood for at least 3 days after HI in mice preimmunized with vtHBsAg but was undetectable in mice preimmunized with wtHBsAg, indicating that vtHBsAgs fail to induce proper immune responses for efficient HBsAg clearance. In conclusion, the biochemical properties of amino acid residues at positions 122 and 145 of HBsAg have a major effect on antigenicity and immunogenicity. In addition, the presence of proper anti-HBs antibodies is indispensable for the neutralization and clearance of HBsAg during HBV infection.     

Escherichia coli DnaA protein: specific biochemical defects of mutant DnaAs reduce initiation frequency to suppress a temperature-sensitive dnaX mutation

The Escherichia coli dnaA73, dnaA721, and dnaA71 alleles, which encode A213D, R432L, T435K substitutions, respectively, were originally isolated as extragenic suppressors of a temperature-sensitive dnaX mutant. As the A213D substitution resides in a domain that functions in ATP binding and the R432L and T435K substitutions affect residues that recognize the DnaA box motif, they might be expected to reduce ATP and specific DNA binding, respectively. Therefore, a major objective was to quantify the biochemical defects of the mutant DnaAs to understand how the altered proteins suppress the temperature-sensitive phenotype of a dnaX mutant. A second purpose was to address the paradox that mutant proteins with substitutions of amino acids essential for recognition of the DnaA box motifs within the E. coli replication origin (oriC) may well be inactive in initiation, yet chromosomal dnaA mutants expressing DnaA proteins with the R432L and T435K substitutions are viable at temperatures from 30 to 39 degrees C. We show biochemically that mutant DnaAs carrying R432L and T435K substitutions fail to bind to the DnaA box sequence. The A213D mutant is sevenfold reduced in its affinity for ATP compared to wild-type DnaA, and its affinity for the DnaA box sequence is also reduced. However, the reduced activity of the A213D mutant in oriC plasmid replication appears to arise from a defect in DnaA oligomerization. Although the T435K mutant fails to bind to the DnaA box sequence, other results suggest that DnaA oligomerization stabilizes the binding of the mutant DnaA to oriC to support its partial activity in initiation in vitro. These results support a model that suppression of dnaX occurs by reducing the frequency of initiation to a manageable level for the mutant DnaX so that viability is maintained.     

球孢白僵菌降解昆虫体壁蛋白酶基因 CDEP-1的克隆与序列分析

构建了球孢白僵菌不同诱导时间的混合cDNA文库。根据丝氨酸类蛋白酶的保守区域设计引物,以构建cDNA文库同样的mRNA为模板,采用RT-PCR法得到长度为594bp的片段BbP。序列测定表明BbP是球孢白僵菌类枯草杆菌蛋白酶Prl的一部分,以BbP为探针,从上述cDNA文库筛选得到长度为1557bp的克隆CDEP-1。CDEP-1含有一个1134bp的开放阅读框（ORF）,编码377个氨基酸,分子量为38616、PI=8.302的蛋白酶前体。CEDP-1的核苷酸序列与蛋白酶K、金龟子绿僵菌Prl、球孢白僵菌Prl的同源性分别为：57.9%、54.7%、83.3%。根据cDNA序列扩增到CDEP-1的基因组序列,分析表明其中含有3个内含子。Southern杂交表明CDEP-1在球孢白僵菌是单拷贝。     

Isolation and characterization of related cDNA clones encoding skeletal muscle beta-tropomyosin and a low-molecular-weight nonmuscle tropomyosin isoform.

We have isolated and characterized cDNA clones from chicken cDNA libraries derived from skeletal muscle, body wall, and cultured fibroblasts. A clone isolated from a skeletal muscle cDNA library contains the complete protein-coding sequence of the 284-amino-acid skeletal muscle beta-tropomyosin together with 72 bases of 5' untranslated sequence and nearly the entire 3' untranslated region (about 660 bases), lacking only the last 4 bases and the poly(A) tail. A second clone, isolated from the fibroblast cDNA library, contains the complete protein-coding sequence of a 248-amino-acid fibroblast tropomyosin together with 77 bases of 5' untranslated sequence and 235 bases of 3' untranslated sequence through the poly(A) tract. The derived amino acid sequence from this clone exhibits only 82% homology with rat fibroblast tropomyosin 4 and 80% homology with human fibroblast tropomyosin TM30nm, indicating that this clone encodes a third 248-amino-acid tropomyosin isoform class. The protein product of this mRNA is fibroblast tropomyosin 3b, one of two low-molecular-weight isoforms expressed in chicken fibroblast cultures. Comparing the sequences of the skeletal muscle and fibroblast cDNAs with a previously characterized clone which encodes the smooth muscle alpha-tropomyosin reveals two regions of absolute homology, suggesting that these three clones were derived from the same gene by alternative RNA splicing.     

DNA sequence of the coding region of the HLA-B44 gene

An HLA-B44 cDNA clone was identified in a cDNA library constructed from an HLA-B44 homozygous cell line. The DNA sequence was determined and was found to contain the complete coding sequence but for (probably) the three N-terminal codons. Comparisons of the derived amino acid sequence with other HLA-A and -B locus amino acid sequences revealed four HLA-B44-specific substitutions including a new polymorphic site. Regions of strong sequence conservation for HLA-B-locus products were found at the nucleotide and amino acid levels.     

Cloning of the human cDNA for the U1 RNA-associated 70K protein.

Anti-RNP sera were used to isolate a cDNA clone for the largest polypeptide of the U1 snRNP, a protein of mol. wt 70 kd designated 70K, from a human liver cDNA library constructed in the expression vector pEX1. The cro-beta-galactosidase-70K fusion protein reacted with various anti-RNP patient sera, a rabbit anti-70K antiserum, as well as with a monoclonal antibody specific for this protein. The sequences of four 70K peptides were determined and they match parts of the deduced amino acid sequence of the 1.3 kb insert of p70.1 indicating that it is a genuine 70K cDNA. Screening of a new cDNA library constructed from polysomal mRNA of HeLa cells with the p70.1 clone yielded an overlapping clone, FL70K, which was 2.7 kb long and covered the complete coding and 3'-untranslated sequence of the 70K protein in addition to 680 nucleotides upstream of the putative initiation codon, The predicted mol. wt of the encoded protein is approximately 70 kd. Amino acid analysis of the purified HeLa 70K protein yielded values close or identical to those deduced from the nucleotide sequence of the full-length cDNA. The 70K protein is rich in arginine (20%) and acidic amino acids (18%). Extremely hydrophilic regions containing mixed-charge amino acid clusters have been identified at the carboxyl-terminal half of the protein, which may function in RNA binding. A sequence comparison with two recently cloned RNA binding proteins revealed homology with one region in the U1 RNP 70K protein. This domain may also be responsible for RNA binding.