Characterization of the ksgA gene of Escherichia coli determining kasugamycin sensitivity

In the plasmid pUC8ksgA7, the coding region of the ksgA gene is preceded by the lac promoter (Plac) and a small open reading frame (ORF). This ORF of 15 codons is composed of nucleotides derived from the lacZ gene, a multiple cloning site and the ksgA gene itself. The reading frame begins with the ATG initiation codon of lacZ and ends a few nucleotides beyond the ATG start codon of ksgA. The ksgA gene is not preceded by a Shine-Dalgarno (SD) signal. Cells transformed with pUC8ksgA7 produce active methylase, the product of the ksgA gene. Introduction of an in-phase TAA stop codon in the small ORF abolishes methylase production in transformed cells. On the plasmid pUC8ksgA5, which contains the entire ksgA region, the promoter of the ksgA gene was found to reside in a 380 base pair Bgl1-Pvu2 restriction fragment, partly overlapping the ksgA gene, by two independent methods. Cloning of this fragment in front of the galK gene in plasmid pKO1 stimulates galactokinase activity in transformants and its insertion into the expression vector pKL203 makes beta-galactosidase synthesis independent of the presence of Plac. The sequence of the Bgl1-Pvu2 fragment was determined and a putative promoter sequence identified. An SD signal could not be distinguished at a proper distance upstream from the ksgA start codon. Instead, an ORF of 13 codons starting with ATG in tandem with an SD signal and ending 4 codons ahead of the ksgA gene was identified. This suggests that translation of the ORF is required for expression of the ksgA gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

A mutation in the latency-related gene of bovine herpesvirus 1 inhibits protein expression from open reading frame 2 and an adjacent reading frame during productive infection

The latency-related (LR) gene of bovine herpesvirus 1 (BHV-1) is abundantly expressed during latency. A mutant BHV-1 strain that contains three stop codons at the 5′ terminus of the LR gene (LR mutant) does not reactivate from latency. This study demonstrates that the LR mutant does not express open reading frame 2 or an adjacent reading frame that lacks an initiating ATG (reading frame C). Since the LR mutant and wild-type BHV-1 express similar levels of LR RNA, we conclude that LR protein expression plays an important role in regulating the latency reactivation cycle in cattle.     

Characterization of a single soybean cDNA encoding cytosolic and glyoxysomal isozymes of aspartate aminotransferase

A soybean cDNA clone, pSAT1, which encodes both the cytosolic and glyoxysomal isozymes of aspartate aminotransferase (AAT; EC 2.6.1.1) was isolated. Genomic Southern blots and analysis of genomic clones indicated pSAT1 was encoded by a single copy gene. pSAT1 contained an open reading frame with ca. 90% amino acid identity to alfalfa and lupin cytosolic AAT and two in-frame start codons, designated ATG1 and ATG2. Alignment of this protein with other plant cytosolic AAT isozymes revealed a 37 amino acid N-terminal extension with characteristics of a peroxisomal targeting signal, designated PTS2, including the modified consensus sequence RL-X₅-HF. The second start codon ATG2 aligned with previously reported start codons for plant cytosolic AAT cDNAs. Plasmids constructed to express the open reading frame initiated by each of the putative start codons produced proteins with AAT activity in Escherichia coli. Immune serum raised against the pSAT1-encoded protein reacted with three soybean AAT isozymes, AAT1 (glyoxysomal), AAT2 (cytosolic), and AAT3 (subcellular location unknown). We propose the glyoxysomal isozyme AAT1 is produced by translational initiation from ATG1 and the cytosolic isozyme AAT2 is produced by translational initiation from ATG2. N-terminal sequencing of purified AAT1 revealed complete identity with the pSAT1-encoded protein and was consistent with the processing of the PTS2. Analysis of cytosolic AAT genomic sequences from several other plant species revealed conservation of the two in-frame start codons and the PTS2 sequence, suggesting that these other species may utilize a single gene to generate both cytosolic and glyoxysomal or peroxisomal forms of AAT.     

Identification of the functional initiation codons of a phase-variable gene of Haemophilus influenzae, lic2A, with the potential for differential expression

Simple sequence repeats located within reading frames mediate phase-variable ON/OFF switches in gene expression by generating frameshifts. Multiple translation initiation codons in different reading frames are found upstream of most Haemophilus influenzae tetranucleotide repeat tracts, raising the possibility of multiple active reading frames and more than two levels of gene expression for these loci. Phase variation between three levels of gene expression (strong, weak, and none) was observed when lic2A was fused to a lacZ reporter gene. The lic2A 5' CAAT repeat tract is preceded by four 5' ATG codons (x, y, z1, and z2) in two reading frames. Each of these initiation codons was inactivated by site-directed mutagenesis. Strong expression from frame 1 was associated with x but not y. Weak expression from frame 2 was mainly dependent on the z2 codon, and there was no expression from frame 3. Using monoclonal antibodies specific for a digalactoside epitope of lipopolysaccharide whose synthesis requires Lic2A, two levels (strong and undetectable) of antibody reactivity were detected, suggesting that weak expression of lic2A is not discernible at the phenotypic level. Inactivation of the x initiation codon resulted in loss of strong expression of the digalactoside epitope and elevated killing by human serum. The failure to detect more than two phenotypes for lic2A, despite clear evidence of weak expression from the z1/z2 initiation codons, leaves open the question of whether or not multiple initiation codons are associated with more complex patterns of phenotypic variation rather than classical phase-variable switching between two phenotypes.     

Efficiencies of translation in three reading frames of unusual non-ORF sequences isolated from phage display.

An unusual nucleotide sequence, called H10, was previously isolated by biopanning with a random peptide library on filamentous phage. The sequence encoded a peptide that bound to the growth hormone binding protein. Despite the fact that the H10 sequence can be expressed in Escherichia coli as a fusion to the gene III minor coat protein of the M13 phage, the sequence contained two TGA stop codons in the zero frame. Several mutant derivatives of the H10 sequence carried not only a stop codon, but also showed frameshifts, either +1 or -1 in individual isolates, between the H10 start and the gene III sequences. In this work, we have subcloned the H10 sequence and three of its derivatives (one requiring a +1 reading frameshift for expression, one requiring a -1 reading frameshift, and one open reading frame) in gene fusions to a reporter beta-galactosidase gene. These sequences have been cloned in all three reading frames relative to the reporter. The non-open reading frame constructs gave (surprisingly) high expression of the reporter (10-40% of control vector expression levels) in two out of the three frames. A site-directed mutant of the TGA stop codon (to TTA) in the +1 shifter greatly reduced the frameshift and gave expression primarily in the zero frame. By contrast, a site-directed mutant of the TGA in the -1 shifter had little effect on the pattern of expression, and alteration of the first TGA (of two) in H10 itself paradoxically reduced expression by half. We believe these phenomena to reflect a translational recoding mechanism in which ribosomes switch reading frames or read past stop codons upon encountering a signal encoded in the nucleotide sequence of the mRNA, because both the open reading frame derivative (which has six nucleotide changes from parental H10) and the site-directed mutant of the +1 shifter, primarily expressed the reporter only in the zero frame.     

天花粉胰蛋白酶抑制剂基因的合成与克隆

天花粉胰蛋白酶抑制剂是从中药天花粉中分离出来的一种新的胰蛋白酶抑制剂,其结构最近被测定为含有三对二硫键的27肽,它也是目前发现的最小的多肽类蛋白酶抑制剂。本文报导了天花粉胰蛋白酶抑制剂基因的合成及克隆。设计的合成基因采用了大肠杆菌偏爱的密码子,全长为108个碱基对,它包括了起始密码子ATG,终止密码子TAG和两端的HindⅢ和BamH Ⅰ的识别顺序。整个基因的合成分为两步,首先用化学方法合成四个DNA片段(F1,38mer;F2,30mer;F3,30mer和F4A,46mer),再经连接酶连接成为两个3′端彼此互补的DNA片段(F1+F2,68mer和F3+F4,76mer),最后从两个互为引物的3′端用Klenow酶聚合补齐得到双链基因。同时,用在基因3′端有两个碱基改变的F4B代替F4A,使基因的终止密码子(TAG)变为甲硫氨酸密码子(ATG),并使基因的阅读框架与质粒pUC19中的LaeZ基因相一致,从而实现该基因与LaeZ基因的融合表达。合成基因经DNA序列分析证明其结构正确。     

Use of chromosomal gene fusions to investigate the role of repetitive DNA in regulation of genes involved in lipopolysaccharide biosynthesis in Haemophilus influenzae.

The lic3 locus of Haemophilus influenzae consists of four open reading frames. The derived amino acid sequences of orf2 and orf4 exhibit homology to Escherichia coli GalE and AdK, respectively. The functions of orf1 and orf3 remain unknown. orf1 contains multiple tandem repeats of the tetrameric DNA sequence CAAT near the 5' end. Two possible translational starts (ATG1 and ATG2) lie upstream. We have used lacZ fusions to investigate whether changes in the number of CAAT repeats in conjunction with differential usage of the upstream frames control the expression of lic3-orf1. Phase-variable expression of lacZ was observed for individual colonies and could be related to variable numbers of CAAT repeats. Of the three possible upstream frames, only one, containing the more downstream of the two possible ATG start codons (ATG2), is used for strong expression of lacZ. Utilization of the more upstream ATG (ATG1) or ATG2 was observed with medium-level expression, while utilization of any of the three possible frames was observed when lacZ was expressed at low to undetectable levels, indicating that other mechanisms may affect expression. To investigate this, lacZ was fused in frame with ATG2 of lic3-orf1, with concomitant deletion of the repeats. Phase-variable expression was still observed, supporting the view that an alternative level of control operates in conjunction with the repeat mechanism.     

Nucleotide sequence and functional analysis of the RAD1 gene of Saccharomyces cerevisiae.

The RAD1 gene of Saccharomyces cerevisiae is involved in excision repair of damaged DNA. The nucleotide sequence of the RAD1 gene presented here shows an open reading frame of 3,300 nucleotides. Two ATG codons occur in the open reading frame at positions +1 and +334, respectively. Since a deletion of about 2.7 kilobases of DNA from the 5' region of the RAD1 gene, which also deletes the +1 ATG and 11 additional codons in the RAD1 open reading frame, partially complements UV sensitivity of a rad1 delta mutant, we examined the role of the +1 ATG and +334 ATG codons in translation initiation of RAD1 protein. Mutation of the +1 ATG codon to ATC affected the complementation ability of the RAD1 gene, whereas mutation of the +334 ATG codon to ATC showed no discernible effect on RAD1 function. These results indicate that translation of RAD1 protein is initiated from the +1 ATG codon. Productive in-frame RAD1-lacZ fusions showed that the RAD1 open reading frame is expressed in yeasts. The RAD1-encoded protein contains 1,100 amino acids with a molecular weight of 126,360.     

Structure of the IgG-binding regions of streptococcal protein G.

The gene encoding the IgG-binding protein G from Streptococcus G148 was isolated by molecular cloning. A subclone containing a 1.5-kb insert gave a functional product in Escherichia coli. Protein analysis of affinity-purified polypeptides revealed two gene products, both smaller than protein G spontaneously released from streptococci, but with identical IgG-binding properties. The complete nucleotide sequence of the insert revealed a repeated structure probably evolved through duplications of fragments of different sizes. The deduced amino acid sequence revealed an open reading frame extending throughout the insert, terminating in a TAA stop codon. Analysis of the two gene products by N-terminal amino acid determination suggests that two different TTG codons are recognized in E. coli for initiation of translation to yield the two products. Based on these results several truncated gene constructions were expressed and analysed. The results suggest that the C-terminal part of streptococcal protein G consists of three IgG-binding domains followed by a region which anchors the protein to the cell surface. Structural and functional comparisons with streptococcal M protein and staphylococcal protein A have been made.     

Chromogenic identification of oligonucleotide-directed mutants.

We describe a simple plaque color assay for identifying oligonucleotide-directed mutations in cloned DNA fragments. The basis of the method is to: fuse the sequence of interest in-frame to the E.coli lacZ gene to produce a blue plaque phage, mutate the site of interest to a stop codon to generate a white plaque phage, and revert the stop codon and surrounding nucleotides to give a blue plaque phage containing one or more desired amino acid changes. The advantages of this cyclic method are that it produces truncated as well as amino acid substituted protein molecules, it can be repeated to introduce additional mutations, and it eliminates the need for labor intensive screening. Essentially any piece of DNA can be mutated using this method if the fragment has one open reading frame. If there is an open reading frame between the site and the lacZ gene, ATG codons can be inserted at the target site. We have used this method to produce termination and amino acid substitution mutants in the yeast CUP1 gene.     

The pAX plasmids: new gene-fusion vectors for sequencing, mutagenesis and expression of proteins in Escherichia coli

A family of plasmid cloning vectors have been constructed, allowing both the sequencing and mutagenesis of foreign genes and the easy isolation of their expression products via fusion proteins in Escherichia coli. Fusion proteins can be inducibly expressed and isolated by affinity chromatography on APTG-Sepharose. The fusion protein consists of beta-galactosidase at the N-terminus, linked by a collagen 'hinge' region containing blood coagulation factor Xa cleavage site to the foreign protein at the C terminus. The factor Xa cleavage site at the N-terminal side of the foreign protein allows the release of the desired amino acid sequence under mild conditions. A multiple cloning site in all three reading frames and stop codons followed by the strong lambda t0 terminator facilitate simple gene insertions and manipulations. The intergenic region of the phage f1 inserted in both orientations allows the isolation of single-stranded DNA from either plasmid-strand for sequencing and mutagenesis. This vector family has been successfully used for the expression and purification of the isoleucyl-tRNA synthetase from Saccharomyces cerevisiae and the histidyl-tRNA synthetase from E. coli.     

Restoration of a translational stop-start overlap reinstates translational coupling in a mutant trpB''-trpA gene pair of the Escherichia coli tryptophan operon.

The trpB and trpA coding regions of the polycistronic trp mRNA of Escherichia coli are separated by overlapping translation stop and start codons. Efficient translation of the trpA coding region is subject to translational coupling, i.e., maximal trpA expression is dependent on prior translation of the trpB coding region. Previous studies demonstrated that the trpA Shine-Dalgarno sequence (within trpB) and/or the location of the trpB stop codon influenced trpA expression. To examine the effect of stop codon location specifically, we constructed plasmids in which different nucleotide sequences preceding the trpA start codon were retained, and only the reading frame was changed. When trpB translation proceeded in the wild type reading frame and terminated at the normal trpB stop codon, trpA polypeptide levels were elevated over the levels observed when translation stopped before or after the natural trpB stop codon. The proximity of the trpB stop codon to the trpA start codon therefore markedly influences trpA expression.     

Possibility of using fusion proteins for detection of nonsense mutations and frame-shift mutations in BRCA1 gene

The aim of this study was to evaluate the possibility of detecting nonsense and frame-shift mutations in exon 11 of brca1 gene by constructing fusion open reading frame (ORF) "exon 11 ORF-alpha-peptide of beta-galactosidase". The ability/inability of this newly constructed ORF to cause alpha-complementation in E. coli delta M15gal cells transformed by the plasmid with the ORF may reflect the absence/presence of nonsense and frame-shift mutations in the studied fragment. A single ORF fragment of exon 11 of brca1 gene--LacZ' gene was designed in pGEN7Zf plasmid, the plasmid was shown to cause Lac+ phenotype in E. coli delta M15gal. Four frame-shift deletion mutations were introduced into exon 11 sequence in the plasmid. Surprisingly, the frame-shift deletion mutations did not influence the ability of plasmids to induce Lac+ phenotype in E. coli delta M15gal in 3 cases and only one deletion mutation resulted in inability of the plasmid to form Lac+ phenotype in E. coli delta M15gal. We suppose that the phenomenon can be explained by the alpha-peptide translation reinitiation from inframe ATG codons situated within the exon 11 sequence. Seven inframe ATG sequences were found in exon 11, at least two in-frame ATG-containing fragments were demonstrated to cause reinitiation. On the other hand, the only deletion mutation resulted in inability of the plasmid to form Lac+ phenotype in E. coli delta M15gal did not leave LacZ' in-frame ATG in econ 11 sequence. We conclude that it is possible to detect frame-shift mutations by in-frame cloning with the LacZ' reporter gene, but this possibility is strongly impeded by the reinitiation of alpha-peptide translation from the in-frame ATG codons within the studied sequence.     

Transport to the cell surface of a peptide sequence attached to the truncated C terminus of an N-terminally anchored integral membrane protein.

Attempts to construct hybrid proteins that are transported to the plasma membrane are frequently unsuccessful because of perturbations in polypeptide folding. In seeking to minimize this problem, we have used the less common type of integral membrane protein, which has an uncleaved signal-anchor domain and an extracellular carboxyl portion, to transport a peptide sequence of interest to the cell surface. A set of plasmids was constructed that contained the gene encoding respiratory syncytial virus glycoprotein G (RSVG) interrupted immediately after one of several proline codons by a synthetic sequence containing unique restriction endonuclease sites and a stop codon. The shortened RSVG gene was flanked by vaccinia virus DNA to permit cloning and expression in a vaccinia virus vector. An open reading frame encoding four copies of the immunodominant repeating epitope of the circumsporozoite protein of Plasmodium falciparum was inserted into the tails of the truncated RSVG genes. Recombinant vaccinia viruses were isolated and shown to express hybrid proteins that reacted with a monoclonal antibody directed to the repeating circumsporozoite epitope. Moreover, immunofluorescence studies indicated that the peptide was on the external cell surface and available to react with antibodies. Expression of the hybrid protein also occurred in rabbits inoculated with the live recombinant vaccinia virus, as demonstrated by the generation of antibodies that bound to P. falciparum sporozoites in vitro.     

Further genetic localization of the transforming sequences of the p21 v-ras gene of Harvey murine sarcoma virus.

The sequences encoding the 21-kilodalton transforming protein (p21 ras) of Harvey murine sarcoma virus have previously been localized genetically to a 1.3-kilobase segment of the viral DNA (E. H. Chang, R. W. Ellis, E. M. Scolnick, and D. R. Lowy, Science 210:1249-1251, 1980). Within this segment, DNA sequence analysis has found a single open reading frame large enough to encode the viral p21 (R. Dhar, R. W. Ellis, T. Y. Shih, S. Oroszlan, B. Shapiro, J. Maizel, D. Lowy, and E. M. Scolnick, Science 217:934-937, 1982). There are three potential in-frame ATG initiation codons at the 5' end of this open reading frame. By constructing a mutant of Harvey murine sarcoma virus DNA from which the first two ATG codons of this open reading frame have been deleted, we now show by transfection of the mutant viral DNA into NIH 3T3 cells that only the third ATG codon is necessary and sufficient for synthesis of the viral p21 and for cellular transformation.     

TGATG vector: a new expression system for cloned foreign genes in Escherichia coli cells

A TGATG vector system was developed that allows for the construction of hybrid operons with partially overlapping genes, employing the effects of translational coupling to optimize expression of cloned cistrons in Escherichia coli. In this vector system (plasmid pPR-TGATG-1), the coding region of a foreign gene is attached to the ATG codon situated on the vector, to form the hybrid operon transcribed from the phage lambda PR promoter. The cloned gene is the distal cistron of this hybrid operon ('overlappon'). The efficiently translated cro'-cat'-'trpE hybrid cistron is proximal to the promoter. The coding region of this artificial fused cistron [the length of the corresponding open reading frame is about 120 amino acids (aa)] includes the following: the N-terminal portions of phage lambda Cro protein (20 aa), the CAT protein of E. coli (72 aa) and 3' C-terminal codons of the E. coli trpE gene product. At the 3'-end of the cro'-cat'-'trpE fused cistron there is a region for efficient translation reinitiation: a Shine-Dalgarno sequence of the E. coli trpD gene and the overlapping stop and start codons (TGATG). In this sequence, the last G is the first nucleotide of the unique SacI-recognition site (GAGCT decreases C) and so integration of the structural part of the foreign gene into the vector plasmid may be performed using blunt-end DNA linking after the treatment of pPR-TGATG-1 with SacI and E. coli DNA polymerase I or its Klenow fragment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide sequence of gene linA encoding resistance to lincosamides in Staphylococcus haemolyticus

We have determined the nucleotide sequence of gene linA in plasmid pIP855, which confers resistance to lincomycin by inactivating it in Staphylococcus haemolyticus. The gene was defined by start and stop codons and an open reading frame of 483 bp corresponding to a product with an Mr of 19020. The apparent size of the resistance-conferring protein was 21 kDa, when a minicell extract was electrophoresed in the sodium dodecyl sulfate-polyacrylamide gels. Full expression of linA was obtained both in Escherichia coli and Bacillus subtilis.