A ‘phase-shift’ fusion system for the regulation of foreign gene expression by lambda represser in Gram-negative bacteria

A ‘phase-shift’ translation fusion vector was constructed in which mutually compatible restriction sites BamHI, BelI and BglII are positioned in such a manner that the cut point is in a different reading frame, immediately following the ATG start codon and ribosome-binding site of the λ cro gene. The λ cro gene is expressed from promoter p_R and controlled by a thermosensitive (cI857) λ repressor. The usefulness of the expression vector was demonstrated using a gal gene lacking the ATG start codon and fusing this to the p_R promoter and ATG start codon of the λ cro gene, resulting in cI857-regulated expression of galactokinase. The vector is of general use for foreign gene expression in Escherichia coli when the target gene has a compatible cohesive end (5′-GATC-3′) at the N terminus (provided, for example, by a BamHI linker). The A λ cI857-p_R-cro-galK cassette was cloned into pJRD215, a wide-host-range plasmid and transferred by conjugation to a variety of Gram-negative bacteria. In all cases, thermosensitive regulation of galactokinase could be demonstrated, though the levels of induction varied considerably. These results show that the powerful λ p_R promoter and the efficient A repressor can be used to regulate expression of foreign genes in Gram-negative organisms other than E. coli.     

A conditional expression vector for the cyanobacteriaSynechocystis sp. strains PCC6803 and PCC6714 orSynechococcus sp. strains PCC7942 and PCC6301

An expression vector, pFC1, has been constructed based on the promiscuous plasmid RSF1010, which provides autonomous replication in several cyanobacteria of the generaSynechocystis andSynechococcus [Mermet-Bouvier et al., Curr Microbiol 26:323–327]. pFC1 harbors the cI857 repressor-encoding gene andp _R promoter, followed by the cro ribosome-binding site and ATG translation initiation codon. The latter is located within the uniqueNdeI restriction site (CATATG) of pFC1 and can be exposed after cleavage with this enzyme for in-frame fusion with the protein-coding sequence to be expressed. TheEscherichia coli lacZ reporter gene cloned in pFC1 appeared to be highly expressed in heat-inducedE. coli or cyanobacterial cells. In every case, -galactosidase amounted to at least 10% of soluble proteins.     

Mechanism of regulating the expression of λN gene by ribosomal protein at translational level

In ribosomal protein S12 mutant or L24 mutant the expression of λN gene was depressed at translational level. To study its mechanism the λN gene region of λN -lacZ gene fusion was trimmed from its 5′ end to 3′ end with DNA exonuclease III (DNA cxoIII) in order to alter the TIR (translational initiation region) and the ding region of λN gene. After DNA sequencing 23 species of different λN-lacZ fused genes were obtained. The β-galactosidase activities of these deletants in ribosomal protein mutant were compared with that in wild type strain. The result indicated that (i) S12 mutant could affect 305 subunit’s binding to the TIR of λN gene messenger and cause the difficulty in forming 30s initiation complex and then decrease the efficiency of translational initiation; (ii) in S12 mutant the coding region of λN gene alw affected the expression λN gene; (iii) in L24 mutant the inhibition of λN gene expression was not related to translational initiation and the 5′ end of the coding region of λN gene, but related to the 3′ end of λN gene.     

甘蔗 UGPase 5’侧翼序列克隆及缺失表达分析简

以甘蔗(FN95;-1702)为材料,通过接头连接PCR方法克隆该基因不同长度5′侧翼序列。将不同长度的5′侧翼序列连同UGPase基因的外显子片段定向插入到GUS基因上游,在保证其后GUS编码框不发生偏移的情况下,插入的UGPase外显子融合GUS表达成为新的报告基因。根据此策略,构建了一系列表达结构为5′ Flanking Sequence-UGPase Exon-GUS-Nos polyA的5′侧翼序列缺失表达载体,进行启动子活性分析。注射法转染烟草叶片组织检测GUS瞬时表达,分析结果表明,所克隆到的UGPase基因5′端侧翼序列不具有启动子活性。     

Pea polyubiquitin genes: (I) structure and genomic organization

Four polyubiquitin genes, PUB1, PUB2, PUB3 and PUB4, were isolated from a pea (Pisum sativum L. cv Alaska) genomic library and completely sequenced. They represent all of the four polyubiquitin genes of the ubiquitin gene family in pea. The coding regions of the genes contain five or six coding units arranged as tandem repeats. The different coding repeats of the four genes share homologies between 75 and 97%, encoding the same protein of 76 amino acids identical to those from other higher plants. The open reading frames of PUB1, PUB2 and PUB4 terminate in the additional amino acid, phenylalanine (F), and PUB3 terminates in isoleucine (I). The polyubiquitin genes all contain intron sequences ranging from 584 to 1114 bp immediately 5′ to the ATG initiation codon of the first coding sequence. Of the four genes, three are associated with long AT-rich (85.4–89.4% A+T) sequences ranging from about 331 to 478 bp at their 5′ or 3′ ends. The PUB4 gene was found to be linked to a moderate to highly repetitive DNA at its 5′ flanking sequence. The greater sequence homology between different genes than among individual repeating units of a gene suggests that the polyubiquitin genes may have arisen by gene duplication of a single gene sequence.     

Kinetics of P22 early gene expression suggests a cro-like regulatory function

Summary Analysis of phage-specified protein synthesis after phage infection of UV-irradiated cells shows a turn-off of early gene expression, a regulatory event that is independent of the known P22 regulatory functions. This supports the suggestion of a cro-like function in P22. We have identified the products of genes 18 and int as contributing to the complex 40,000 dalton band in our SDS-polyacrylamide gels. Both gene products appear to be subject to regulation by the cro-like function of P22. Proteins of 33,000, 29,000, 27,000, 25,000, and 24,000 MW, specified by as yet unidentified P22 genes of the early leftward operon, are regulated by the same function. Our data suggest that the cro-like function is expressed from the early rightward operon.     

Vectors for regulated high-level expression of proteins fused to truncated forms of Escherichia coli β-galactosidase

Vectors were constructed that allow the expression of large quantities of fused proteins in Escherichia coli. The plasmids carry the E. coli lac UV5 promotor and different portions of the coding sequence of the E. coli lacZ gene. The truncated lacZ genes are flanked by a polylinker region either at their 5′ end or their 3′ end, which has several unique restriction sites. Thus, coding sequences of any prokaryotic and eukaryotic gene can be ligated in frame to the truncated lacZ genes. These vectors were used for high-level production of herpes simplex virus type 1 envelope glycoprotein D antigens.     

Kinetics of bacteriophage lambda repressor synthesis directed by the PRE promoter: influence of temperature, multiplicity of infection, and mutation of PRM or the cro gene

Summary Expression of the P _RE (establishment) pathway for repressor synthesis is regulated both by phage-specific genetic elements and by physiological conditions. Here we describe the effects of temperature, multiplicity of infection, mutations in the cro gene, and a mutation in P _RM on P _RE-directed repressor synthesis. As Reichardt (1975a) has shown, repressor synthesis begins 5–15 min after infection by wildtype phage, and is shut off at 20–30 min after infection, depending on the temperature. At 43°, synthesis starts sooner, shuts off earlier, and leads to lower repressor levels than are attained at lower temperatures. Experiments with the temperature sensitive mutant crots20 demonstrate that, as had been shown previously in experiments at 30° and 37° C, cro protein is responsible for the shut-off of repressor synthesis at 43°. In addition to the effects of temperature, the kinetics of repressor synthesis are strongly affected by multiplicity of infection (moi). At mois greater than 10, repressor synthesis after infection by wildtype at 30° is dramatically inhibited. Unexpectedly, the P _RM mutation prm116, under certain conditions, can alleviate both cro-mediated shutoff and the inhibition of P _RE-directed repressor synthesis at high moi. These effects of prm116 are observed only at low temperature (30°–32° C) and at mois of about 6–10 or greater; they also appear to be cis-specific. Possible mechanisms for the effects of the prm116 mutation are discussed. Finally, these studies demonstrate that crots20, which was isolated as a temperature-sensitive lethal mutation in the cro gene (Herskowitz, unpublished), is temperature-sensitive with respect to the ability to shutoff P _RE-directed repressor synthesis; however, even at low temperature (30° C), the crots20 gene product is only partially active.