Photosystem II of rye. Cloning and determination of the nucleotide sequence of chloroplast DNA fragments, comprising the psbA gene coding for D1 proteins

EcoRI and BamHI fragments of rye chloroplast DNA comprising psbA gene were cloned and a 2729 bp region was sequenced. Cloning of EcoRI fragment into pTZ19R plasmid led to a single nucleotide deletion in the coding region of psbA gene. A scheme of full-length psbA gene cloning is proposed, allowing one to escape the damage effect of the psbA gene expression product on the host cell. The differences between monocot and dicot in nucleotide sequences of DNA downstream of psbA genes are discussed. Gene rps19 is located 131 bp downstream from psbA gene on the complementary strand. The amino acid sequences of D1 and S19 proteins of different species are compared.     

Escherichia coli plasmid vectors containing synthetic translational initiation sequences and ribosome binding sites fused with the lacZ gene

The construction of a series of Escherichia coli plasmid vectors suitable for assaying the effects of gene control signals fused with the E. coli lacZ gene is reported. A synthetic deoxyoligonucleotide dodecamer 5'-CATGAATTCATG GTACTTAAGTAC-5' containing two translation initiation codons (ATG) separated by an EcoRI site was ligated with a lacZ gene derivative which lacks the codons for the first eight amino acids in plasmid pMC1403 (Casadaban et al., 1980). Two ribosome-binding sequences were synthesised and inserted into the EcoRI site before an ATG, and the effects of these sequences on lacZ gene expression in vivo measured by assaying beta-galactosidase activity. The E. coli ribosomal RNA gene (rrnB) promoter, the tetracycline resistance gene promoter, and a lambda phage promoter were cloned using these plasmids. The plasmids are 9.9 kb in size, have ampicillin resistance as a selectable marker and are generally useful for the detection and in vivo assay of gene control regions.     

重组人血小板生成素在大肠杆菌中表达的研究

采用化学法全合成了编码人血小板生成素（ｔｈｒｏｍｂｏｐｏｉｅｔｉｎ,ＴＰＯ）成熟肽Ｎ端１５３氨基酸的基因序列,构建基于该合成基因的表达质粒,结果以谷胱甘肽转硫酶－ＴＰＯ１５３（ＧＳＴ－ＴＰＯ１５３）融合蛋白的方式获得了占全菌蛋白４０％的高效表达．进一步采用ＰＣＲ方法分别对ＴＰＯ合成基因及ＴＰＯｃＤＮＡ的翻译起始区（ＴＩＲ）序列进行定点突变,以降低这一区域的Ｇ－Ｃ含量．将突变序列分别插入到ｐＢＶ２２０表达载体中,重组质粒在转化大肠杆菌ＪＭ１０９后,均获得了表达,其中ＴＩＲ区突变后的合成基因表达产物约占全菌蛋白的１５％．为研究基因下游结构对表达的影响,在不改变氨基酸组成的基础上,构建了ＴＰＯ合成基因与ＴＰＯｃＤＮＡ的杂合序列表达质粒．研究结果表明翻译起始效率是影响ｒｈ－ＴＰＯ在大肠杆菌中表达的重要因素之一,同时基因下游序列的组成对表达水平也会产生影响．     

Cloning, sequencing, and expression of thermophilic Bacillus sp. strain TB-90 urease gene complex in Escherichia coli.

The urease of thermophilic Bacillus sp. strain TB-90 is composed of three subunits with molecular masses of 61, 12, and 11 kDa. By using synthetic oligonucleotide probes based on N-terminal amino acid sequences of each subunit, we cloned a 3.2-kb EcoRI fragment of TB-90 genomic DNA. Moreover, we cloned two other DNA fragments by gene walking starting from this fragment. Finally, we reconstructed in vitro a 6.2-kb DNA fragment which expressed catalytically active urease in Escherichia coli by combining these three DNA fragments. Nucleotide sequencing analysis revealed that the urease gene complex consists of nine genes, which were designed ureA, ureB, ureC, ureE, ureF, ureG, ureD, ureH, and ureI in order of arrangement. The structural genes ureA, ureB, and ureC encode the 11-, 12-, and 61-kDa subunits, respectively. The deduced amino acid sequences of UreD, UreE, UreF, and UreG, the gene products of four accessory genes, are homologous to those of the corresponding Ure proteins of Klebsiella aerogenes. UreD, UreF, and UreG were essential for expression of urease activity in E. coli and are suggested to play important roles in the maturation step of the urease in a co- and/or posttranslational manner. On the other hand, UreH and UreI exhibited no significant similarity to the known accessory proteins of other bacteria. However, UreH showed 23% amino acid identity to the Alcaligenes eutrophus HoxN protein, a high-affinity nickel transporter.     

Expressing plasmid vectors on the basis of Escherichia coli beta-galactosidase gene fragments

With the use of synthetic DNA fragments, a set of new plasmid vectors has been obtained. The vectors provided high level expression of peptides and small proteins in E. coli as fusions with fragments of beta-galactosidase of various length. These vectors were used to achieve expression of a synthetic gene for a functionally active fragment of bacteriorhodopsin. The yields of hybrid proteins consisting of beta-galactosidase and bacteriorhodopsin fragments were in the range of 5-30% from the total amount of cellular protein.     

Purification of two chitinases from Rhizopus oligosporus and isolation and sequencing of the encoding genes.

Two chitinases were purified from Rhizopus oligosporus, a filamentous fungus belonging to the class Zygomycetes, and designated chitinase I and chitinase II. Their N-terminal amino acid sequences were determined, and two synthetic oligonucleotide probes corresponding to these amino acid sequences were synthesized. Southern blot analyses of the total genomic DNA from R. oligosporus with these oligonucleotides as probes indicated that one of the two genes encoding these two chitinases was contained in a 2.9-kb EcoRI fragment and in a 3.6-kb HindIII fragment and that the other one was contained in a 2.9-kb EcoRI fragment and in a 11.5-kb HindIII fragment. Two DNA fragments were isolated from the phage bank of R. oligosporus genomic DNA with the synthetic oligonucleotides as probes. The restriction enzyme analyses of these fragments coincided with the Southern blot analyses described above and the amino acid sequences deduced from their nucleotide sequences contained those identical to the determined N-terminal amino acid sequences of the purified chitinases, indicating that each of these fragments contained a gene encoding chitinase (designated chi 1 and chi 2, encoding chitinase I and II, respectively). The deduced amino acid sequences of these two genes had domain structures similar to that of the published sequence of chitinase of Saccharomyces cerevisiae, except that they had an additional C-terminal domain. Furthermore, there were significant differences between the molecular weights experimentally determined with the two purified enzymes and those deduced from the nucleotide sequences for both genes. Analysis of the N- and C-terminal amino acid sequences of both chitinases and comparison of them with the amino acid sequences deduced from the nucleotide sequences revealed posttranslational processing not only at the N-terminal signal sequences but also at the C-terminal domains. It is concluded that these chitinases are synthesized with pre- and prosequences in addition to the mature enzyme sequences and that the prosequences are located at the C terminal.     

High-level expression of RNAs and proteins: the use of oligonucleotides for the precise fusion of coding-to-regulatory sequences

We show that the fusion between regulatory sequences present on expression vectors and coding sequences can be efficiently achieved by oligonucleotide-directed mutagenesis. We have constructed single-stranded (ss) expression vectors that facilitate this process. These plasmids derive from vectors that have been used for the synthesis of quantities of proteins in Escherichia coli or RNAs in vitro. By inserting the origin of replication of the ss phage f1 into these plasmids it became possible to package their ss DNA into phage rods. Deletion of unwanted sequences or simple base changes can then be obtained by oligonucleotide-directed mutagenesis using the vector ss DNA as a template. We discuss the results of several experiments where this technique was applied to our expression vectors and we demonstrate the construction of a plasmid which efficiently synthesizes in vitro a regulatory RNA molecule that is involved in the control of plasmid copy number.     

Generation of mammalian cells stably expressing multiple genes at predetermined levels

Expression of cloned genes at desired levels in cultured mammalian cells is essential for studying protein function. Controlled levels of expression have been difficult to achieve, especially for cell lines with low transfection efficiency or when expression of multiple genes is required. An internal ribosomal entry site (IRES) has been incorporated into many types of expression vectors to allow simultaneous expression of two genes. However, there has been no systematic quantitative analysis of expression levels in individual cells of genes linked by an IRES, and thus the broad use of these vectors in functional analysis has been limited. We constructed a set of retroviral expression vectors containing an IRES followed by a quantitative selectable marker such as green fluorescent protein (GFP) or truncated cell surface proteins CD2 or CD4. The gene of interest is placed in a multiple cloning site 5' of the IRES sequence under the control of the retroviral long terminal repeat (LTR) promoter. These vectors exploit the approximately 100-fold differences in levels of expression of a retrovirus vector depending on its site of insertion in the host chromosome. We show that the level of expression of the gene downstream of the IRES and the expression level and functional activity of the gene cloned upstream of the IRES are highly correlated in stably infected target cells. This feature makes our vectors extremely useful for the rapid generation of stably transfected cell populations or clonal cell lines expressing specific amounts of a desired protein simply by fluorescent activated cell sorting (FACS) based on the level of expression of the gene downstream of the IRES. We show how these vectors can be used to generate cells expressing high levels of the erythropoietin receptor (EpoR) or a dominant negative Smad3 protein and to generate cells expressing two different cloned proteins, Ski and Smad4. Correlation of a biologic effect with the level of expression of the protein downstream of the IRES provides strong evidence for the function of the protein placed upstream of the IRES.     

Bacteriophage lambda and plasmid vectors, allowing fusion of cloned genes in each of the three translational phases.

We have constructed vectors from bacteriophage lambda and from plasmid pBR322 having a single EcoRI restriction site which is immediately downstream from the lac UV5 promotor. Each vector allows the fusion of a cloned gene to the lac Z gene in a different phase relative to the translation initiation codon of the lac Z gene. These vectors were constructed through modification of the initial EcoRI restriction site by S1 endonuclease treatment and then addition of octadeoxyribonucleotides (EcoRI linkers), which shifted the restriction site by 2 or 4 nucleotides. Used in combination these vectors should allow translation of a cloned gene in any one of the three coding phases. The bacteriophages vectors are certified as B2 (EK2) safety level vectors by the French "recombinaison génétique in vitro" committee (D.G.R.S.T.).     

High-level expression vectors to synthesize unfused proteins in Escherichia coli

A new class of plasmid vectors (pANK-12, pANH-1, and pPL2) for synthesizing unfused proteins was constructed by inserting synthetic linkers at the NdeI site (CATATG) of plasmid pJL6, which contains the lambda cII gene initiator codon. These expression vectors contain the lambda pL promoter, the cII ribosome-binding site, cII start codon and unique restriction sites (KpnI, Asp718, HpaI, BamHI) downstream from the initiator ATG for expression of unfused proteins. The main advantage of these vectors is that any DNA fragment with an open reading frame that does not possess a start and/or a stop codon can be directed to overproduce protein in an unfused form.     

Molecular cloning and deletion of the gene encoding aspergillopepsin A from Aspergillus awamori

We have cloned genomic pepA sequences encoding the aspartic proteinase aspergillopepsin A (PEPA) from Aspergillus awamori using a synthetic oligodeoxyribonucleotide probe. Nucleotide sequence data from the pepA gene revealed that it is composed of four exons of 320, 278, 249, and 338 bp. Three introns which interrupt the coding sequence are 51, 52, and 59 bp in length. Directly downstream from the putative start codon lies a sequence encoding 69 amino acids (aa) which are not present in mature PEPA. Based on similarities to other aspartic proteinases, this region may represent a 20-aa signal peptide followed by a 49-aa propeptide that is rich in basic aa residues. Northern blots of total cellular RNA extracted from A. awamori cells indicate that pepA is transcribed as a single 1.4-kb mRNA. Mutants of A. awamori lacking the pepA structural gene were derived by the following gene replacement strategy. First, we constructed a plasmid in which a 2.4-kb SalI fragment containing the entire pepA coding region was deleted from a 9-kb Eco RI genomic DNA clone and replaced by a synthetic DNA polylinker. Second, a selectable argB gene was inserted into the polylinker. Third, the EcoRI fragment which contained the argB marker flanked by pepA sequences was excised from the plasmid and used to transform an argB auxotroph of A. awamori. From 16-40% of the resulting prototrophic transformants were found to have a PEPA-deficient phenotype when screened with an immunoassay using antibodies specific for PEPA. Southern hybridization experiments confirmed that these mutants resulted from a gene replacement event at the pepA locus.     

Choice of expression vector alters the localization of a human cellular protein

The fusion of synthetic epitopes with proteins of interest is an important tool in the identification and characterization of recombinant proteins. Several mammalian expression vectors are commercially available containing unique identification tags or epitopes. These vectors offer a great advantage to researchers, as highly specific antibodies and purification resins against these specific epitopes are readily available. The tags facilitate immunologic assays and the purification of the recombinant proteins. The fusion of these epitopes with the recombinant proteins is not expected to alter the behavior of the protein of interest. In this report, we demonstrate that the mere expression of a cellular protein, hVIP/mov34, which we earlier identified as a cellular HIV-1 Vpr ligand, in two different vectors clearly altered its localization pattern in HeLa cells. Specifically, cloning of hVIP/mov34 in pcDNA3/HisA resulted in its nuclear localization, whereas the expression of this gene from a TOPO cloning expression vector, pcDNA3.1/V5/His, resulted in cytoplasmic expression. The native staining pattern of hVIP/mov34 using polyclonal antisera raised against hVIP/mov34 demonstrated cytoplasmic staining. During cloning, other leader sequences intended for targeting this protein into a cytoplasmic or a nuclear location were not fused to the actual ORF of this protein. Also, the amino acid sequence of the fusion region arising from cloning of hVIP/mov34 in both vectors does not match any reported NLS sequences. These results indicate that the choice of the expression vectors, as well as the position of synthetic epitopes, can significantly alter the behavior and the biology of recombinant proteins. This result suggests the need for a careful examination of these features when characterizing a newly identified protein.     

Characterization and regulation of Schizosaccharomyces pombe gene encoding thioredoxin

A cDNA coding thioredoxin (TRX) was isolated from a cDNA library of Schizosaccharomyces pombe by colony hybridization. The 438 bp EcoRI fragment, which was detected by Southern hybridization, reveals an open reading frame which encodes a protein of 103 amino acids. The genomic DNA encoding TRX was also isolated from S. pombe chromosomal DNA using PCR. The cloned sequence contains 1795 bp and encodes a protein of 103 amino acids. However, the C-terminal region obtained from the cDNA clone is -Val-Arg-Leu-Asn-Arg-Ser-Leu, whereas the C-terminal region deduced from the genomic DNA appears to contain -Ala-Ser-Ile-Lys-Ala-Asn-Leu. This indicates that S. pombe cells contain two kinds of TRX genes which have dissimilar amino acid sequences only at the C-terminal regions. The heterologous TRX 1C produced from the cDNA clone could be used as a subunit of T7 DNA polymerase, while the TRX 1G from the genomic DNA could not. The upstream sequence and the region encoding the N-terminal 18 amino acids of the genomic DNA were fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357 to generate the fusion plasmid pYKT24. Synthesis of beta-galactosidase from the fusion plasmid was found to be enhanced by hydrogen peroxide, menadione and aluminum chloride. It indicates that the expression of the cloned TRX gene is induced by oxidative stress.     

Secretion cloning vectors in Escherichia coli

The DNA fragment coding for the signal peptide of the OmpA protein, a major outer membrane protein of Escherichia coli, has been inserted into the high-level expression vectors, pIN-III. A foreign DNA fragment can be cloned in any one of the three reading frames at the unique EcoRI, HindIII or BamHI sites immediately after the ompA signal peptide coding sequence. The cloned foreign gene is under the control of both the lpp promoter and the lac promoter-operator. The expression of the gene is regulated by the lac repressor produced by the same vectors. Using the pIN-III-ompA vector, the DNA fragment coding for only the mature portion of beta-lactamase was inserted into the EcoRI site. Upon induction of gene expression, beta-lactamase was secreted into the periplasmic space. The ompA signal peptide was correctly removed resulting in the production of beta-lactamase with four extra amino acid residues (Gly-Ile-Pro-Gly) at its amino terminus due to the linker sequence in the vector. After a 3-h induction, beta-lactamase was accumulated to 20% of total cellular protein without any detectable accumulation of pro-beta-lactamase. Using oligonucleotide-directed site-specific mutagenesis, we have also removed the linker sequence and upon induction of gene expression, beta-lactamase with the authentic NH2-terminal sequence was produced, in even larger amounts than the beta-lactamase with the linker sequence.     

A new series of trpE vectors that enable high expression of nonfusion proteins in bacteria.

Expression of recombinant proteins in bacteria has facilitated the characterization of many gene products. However, the biochemical characterization of recombinant proteins is limited since the bacterially expressed proteins are often synthesized as fusion polypeptides. The presence of bacterial sequences in fusion proteins further limits the use of these proteins for generating antibodies since the bacterial sequences are also antigenic. We describe two new bacterial expression vectors based on the pATH series of plasmids. These vectors were made by precisely deleting all of the trpE coding sequences found in pATH. The new vectors have enabled us to express eukaryotic genes as nonfusion polypeptides. These altered plasmids can be used to insert any DNA sequence of interest through a multiple cloning site located just 3' of an ATG start codon. Protein expression is still under the control of the trp operon and is carried out at great efficiency when the bacteria are tryptophan deprived. Studies presented here test the expression system with neurofilament subunits, NF-L and NF-H. Large amounts of recombinant nonfusion proteins were produced. Also, a time course of induction shows that the production of the nonfusion proteins was under the control of the trp operon which is readily inducible after tryptophan starvation and addition of indoleacrylic acid. These vectors may be useful for the overexpression of many proteins in a form closely approximating their native state.     

Polypeptide sequences involved in the cleavage of DNA by the restriction endonuclease EcoRI

We have prepared a variety of fragments of the restriction endonuclease EcoRI by partial or total CNBr or acid cleavage of the protein. These fragments were isolated by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. They were analyzed in a qualitative manner for phosphodiesterase activity. Antibodies against these fragments were elicited in rats and tested for binding to native EcoRI in an enzyme-linked immunoassay. We conclude from these experiments that the DNA binding site of EcoRI is located in the COOH-terminal half of the molecule, close to and probably comprising amino acid residues 137 to 157. This conclusion is reinforced by the observation that this sequence shows homology to the sequences of the recognition helix of other gene-regulatory proteins.     

Vectors for expression of truncated coding sequences in Escherichia coli

We describe the construction of vectors for expressing in Escherichia coli DNA fragments obtained by progressive deletions of DNA inserts in single-stranded sequencing vectors as M13 or pTZ according to the methode of Dale et al. (Plasmid 1985, 13, 31-40). These vectors, pIMS1, pIMS5, and pIMS6, harbor all of the elements required for the regulated expression of any open reading frame flanked by EcoRI restriction sites. The encoded peptides contain only a few vector-derived amino acids. A method is described for direct selection of recombinant clones by in situ RNA hybridization. The properties of the expression vector have been analyzed with a DNA deletion series obtained from the cDNA coding for the regulatory subunit of Dictyostelium discoideum cAMP-dependent protein kinase.