Gene expression in Deinococcus radiodurans.

We previously reported that the Escherichia coli drug-resistance determinants aphA (kanamycin-resistance) and cat (chloramphenicol-resistance) could be introduced to Deinococcus radiodurans by transformation methods that produce duplication insertion. However, both determinants appeared to require dramatic chromosomal amplification for expression of resistance. Additional studies described here, confirming this requirement for extensive amplification, led us to the use of promoter-probe plasmids in which the E. coli promoter has been deleted, leaving only coding sequences for the marker gene. We find that the insertion of D. radiodurans sequences immediately upstream from the promoterless drug-resistance determinant produces drug-resistant transformants without significant chromosomal amplification. Furthermore, a series of stable E. coli-D. radiodurans shuttle plasmids was devised by inserting fragments of D. radiodurans plasmid pUE10 in an E. coli plasmid directly upstream from a promoterless cat gene. These constructions replicated in D. radiodurans by virtue of the pUE10 replicon and expressed the cat determinant because of D. radiodurans promoter sequences in the pUE10 fragment. Of three such constructions, none expressed the cat gene in E. coli. Similar results were obtained using a promoterless tet gene. Translational fusions were made between D. radiodurans genes and E. coli 5'-truncated lacZ. Three fusions that produced high levels of beta Gal in D. radiodurans were introduced into E. coli, but beta Gal was produced in only one. The results demonstrate that the E. coli genes cat, tet and lacZ can be efficiently expressed in D. radiodurans if a D. radiodurans promoter is provided, and that D. radiodurans promoters often do not function as promoters in E. coli.     

Isolation and characterization of sporulation-specific promoters in the yeast Saccharomyces cerevisiae

A library of random yeast genomic DNA:lacZ fusions has been constructed using an episomal yeast-Escherichia coli shuttle vector (pCS1). Plasmid pCS1 requires insertion of a promoter and an in frame ATG codon upstream of its resident truncated lacZ gene to regulate expression in yeast. Yeast genomic DNA fragments of 4-6 kb were generated by partial digestion with Sau3A and ligated into the unique BamHI site of plasmid pCS1 to generate a library of 5 x 10(4) individual E. coli transformants. This library was screened to identify promoter-lacZ fusions that were expressed uniquely during sporulation. Of 342 yeast transformants that exhibited beta-galactosidase activity, two were found to express the lacZ gene in a sporulation-specific manner. This paper presents the characterization of two genomic yeast DNA fragments containing promoters that control lacZ expression during the sporulation process. Expression from the promoter present in plasmid pJC18 occurred from 11-21 hours into the sporulation process, while the promoter in plasmid pJC217 was active from 4-14 hours. Staining of nuclear DNA to correlate nuclear morphology with timing of gene expression showed when each of these promoters was active in terms of the morphological stages of sporulation.     

A family of yeast expression vectors containing the phage f1 intergenic region

The construction and characterization of a family of yeast expression vectors is described. They have the following features: plasmid replication and selection (ApR) in Escherichia coli, packaging of single-stranded (ss) DNA upon infection of E. coli with a filamentous helper phage, replication in Saccharomyces cerevisiae based on the 2 mu plasmid origin of replication (ori), selection in yeast by complementation of LEU2 (pVT-L series, size 6.3 kb) or URA3 gene (pVT-U series, size 6.9 kb) and seven unique restriction sites for cloning within an 'expression cassette' which includes the promoter and 3' sequence of the ADH1 gene. The multiple cloning site as well as the ori and intergenic region of the phage f1 have been cloned in two orientations for convenient gene cloning and ssDNA strand selection. As a result any of these eight vectors can be chosen for cloning, expressing genes in yeast, sequencing and mutagenesis without the need for recloning into specialized vectors.     

In vivo functional characterization of a yeast nucleotide sequence: construction of a mini-Mu derivative adapted to yeast

We have constructed a derivative of the bacteriophage Mu (called MudIIZZ1), which contains the lacZ gene coding for beta-galactosidase (beta Gal) and markers suited for yeast transformation (2 mu circle replication origin and LEU2). This new transposon is an efficient tool for studying the expression of cloned yeast nucleotide sequences through beta Gal-protein fusions. It is also adapted for one-step disruption experiments so that a functional map of the same sequence can be drawn. We have used this MudIIZZ1 transposon to study a 5-kb DNA fragment which had been cloned by complementation of a cold-sensitive respiration-deficient phenotype. By testing the expression of the beta Gal fusions and the disruption phenotype, we have confirmed the presence of a gene required for mitochondrial functions, and revealed another two open reading frames in the same fragment; one of these also interferes with mitochondrial biogenesis. The method is fast and reliable, and has potential for more general purposes which are discussed.     

A vector for construction of gene libraries and the expression of heterologous genes in Saccharomyces cerevisiae

We have constructed a convenient new vector, YEp-DE, for the construction of gene libraries and the expression of heterologous genes in Saccharomyces cerevisiae. The vector contains the yeast LEU2 gene, the 2 mu origin of replication, and a region from pUC18 that includes the ampr gene, the Escherichia coli origin of replication (ori), and the LacZ gene with multiple cloning sites. Five sites (Sac1, Sma1, BamH1, Sal1, Sph1) in this region are unique. This vector has advantages over similar yeast-E. coli shuttle vectors: small size (7291 bp, entirely sequenced), convenient cloning sites, and lacZ selection for detecting recombinant plasmids.     

A modular set of lacZ fusion vectors for studying gene expression in Caenorhabditis elegans

We describe a series of plasmid vectors which contain modular features particularly useful for studying gene expression in eukaryotic systems. The vectors contain the Escherichia coli beta-galactosidase (beta Gal)-encoding region (the lacZ gene) flanked by unique polylinker segments on the 5' and 3' ends, and several combinations of a variety of modules: a selectable marker (an amber suppressor tRNA), a translational initiation region, a synthetic intron segment, the early polyadenylation signal from SV40, and 3' regions from two nematode genes. A segment encoding the nuclear localization peptide from the SV40 T antigen is incorporated into many of the constructs, leading to beta Gal accumulation in nuclei, which can facilitate identification of producing cells in complex tissues. To make functional beta Gal fusions to secreted proteins, we constructed plasmids with an alternate module encoding a synthetic transmembrane domain upstream from lacZ. This domain is designed to stop transfer of secreted proteins across the membrane during secretion, allowing the beta Gal domain of the fusion polypeptide to remain in the cytoplasm and thus function in enzymatic assays. We have used the vectors to analyze expression of several genes in the nematode Caenorhabditis elegans, and have demonstrated in these studies that lacZ can be expressed in a wide variety of different tissues and cell types. These vectors should be useful in studying gene expression both in C. elegans and in other experimental systems.     

Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis

A single-copy integration vector was used for the in vitro construction of translational fusions to the lacZ gene of Escherichia coli. Insertion of a single copy of the lacZ fusion into the B. subtilis chromosome leads to an easily detected Amy- phenotype. A trpE-lacZ fusion was constructed in which the trp promoter directs hybrid beta-galactosidase (beta Gal) synthesis. The level of beta Gal in a wild-type strain carrying the trpE-lacZ fusion in the chromosome is regulated by exogenous tryptophan, while a 5-methyltryptophan-resistant mutant constitutively synthesizes betaGal. A trpF-lacZ fusion was constructed and used to determine the effect of a frameshift mutation in the trpE gene on expression of the trpF-lacZ fusion. The frameshift mutation in trpE led to a three-fold reduction in the levels of the trpF-lacZ fusion. The levels of the betaGal activity of these integrated lacZ fusions appear to provide a quantitative measure of the expression of B. subtilis genes under single-copy conditions.     

Candida glabrata shuttle vectors suitable for translational fusions to lacZ and use of beta-galactosidase as a reporter of gene expression

The functionality of beta-galactosidase encoded by the E. coli lacZ gene as a reporter of gene expression in C. glabrata was investigated. C. glabrata/E. coli shuttle vectors were constructed, containing both a C. glabrata CEN-ARS cassette, to allow regular segregation and episomal replication of the plasmids, and the lacZ coding sequence of E. coli. The functionality of beta-galactosidase in C. glabrata was verified by inserting the promoter and the 5' coding region of the HIS3 gene from C. glabrata directionally upstream of the lacZ gene. By fusing the promoter of the copper-controlled MTII gene to the lacZ reporter, we showed that beta-galactosidase activity can be differentially induced in C. glabrata. beta-galactosidase reporter activities were detected qualitatively by an indirect filter assay and quantitatively from permeabilized cells.     

Cloning and characterization of a novel beta-galactosidase-coding gene from Rhizobium meliloti

The Rhizobium meliloti (Rm) lacZ gene provides a convenient model to investigate patterns of gene regulation in these agronomically important bacteria. A gene encoding beta-galactosidase (beta Gal) activity was cloned from R. meliloti by complementing a lactose-negative Escherichia coli mutant. A series of Sau3A subclones was generated in pBR322, and the coding region for the beta Gal-coding gene was localized to a 2.4-kb core fragment. In E. coli 'maxicells', these lacZ subclones produced a 79-kDa polypeptide, irrespective of the fragment size demonstrating that the translation initiation signal(s) are located on the 2.4-kb fragment. Transposon Tn5 mutagenesis and BAL 31 deletion analysis showed that the expression of the Rm lacZ gene in E. coli was dependent on the tetracycline-resistance promoter of pBR322. The cloned sequence was required for beta Gal synthesis in Rhizobium since mutants generated by reverse genetics lack this enzyme and were specifically defective in lactose catabolism.     

Synthesis of maize chloroplast ATP-synthase beta-subunit fusion proteins in Escherichia coli and binding to the inner membrane

The maize chloroplast gene for the beta subunit (atpB) of the chloroplast CF1 component of ATPase from maize, when fused to either the lacZ or ral genes in the vectors pMC1403 or pHUB4, is expressed in Escherichia coli as a fusion protein with beta-galactosidase or with bacteriophage lambda Ral sequences. Some of the fusion proteins are converted to a membrane-bound form, as determined by differential and sucrose-gradient centrifugation. The specificity of membrane binding has been examined using E. coli unc mutants that are defective in binding of the F1 ATPase component to the F0 receptor site on the membrane, and by the use of two different length maize atpB::lacZ gene fusions. We show that the first 365 N-terminal amino acids (aa) of the maize beta subunit are involved in binding to the E. coli inner membrane, and that this binding is probably mediated by the bacterial F0 receptor.     

Two versatile shuttle vectors for Thermus thermophilus-Escherichia coli containing multiple cloning sites, lacZα gene and kanamycin or hygromycin resistance marker

Two versatile shuttle vectors for Thermus thermophilus and Escherichia coli were developed on the basis of the T. thermophilus cryptic plasmid pTT8 and E. coli vector pUC13. These shuttle vectors, pTRK1T and pTRH1T, carry a gene encoding a protein homologous to replication protein derived from pTT8, a replicon for E. coli, new multiple cloning sites and a lacZα gene from E. coli vector pUC13, and also have a gene encoding a thermostable protein that confers resistance to kanamycin or hygromycin, which can be used as a selection marker in T. thermophilus. These shuttle vectors are useful to develop enzymes and proteins of biotechnological interest. We also constructed a plasmid, pUC13T, which carries the same multiple cloning sites of pTRK1T and pTRH1T. These vectors should facilitate cloning procedures both in E. coli and T. thermophilus.     

Expression vector system based on the chicken beta-actin promoter directs efficient production of interleukin-5

We examined the promoter activity of the 1.3-kb chicken beta-actin gene sequence located between the 5' flanking region and the proximal region of the second exon. This promoter region showed higher promoter activity than the simian virus 40 (SV40) early promoter or the Rous sarcoma virus (RSV) long terminal repeat (LTR) as assayed by transient lacZ gene expression in mouse L cells. Furthermore, replacement of the 3' splice sequence in this promoter by that derived from the rabbit beta-globin gene resulted in a approximately 2.5-fold enhancement in the synthesis of beta-galactosidase (beta Gal). Introduction of the SV40 origin of DNA replication (ori) into the vector carrying this hybrid promoter, which we designate the AG promoter, markedly enhanced the production of beta Gal in an SV40 T antigen-producing cell, BMT10. We have constructed a useful vector containing the strong AG promoter, several unique restriction sites, a SV40 polyadenylation signal and the SV40 ori for transient expression of cDNA in BMT10 or COS cells. We demonstrate the use of this vector for efficient production of interleukin-5 in BMT10 cells.     

New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments

Two new shuttle vectors have been constructed by fusing the Escherichia coli plasmid pUC9 with the Staphylococcus aureus plasmids pU110 and pC194. The resulting hybrids replicate in both E. coli and Bacillus subtilis and contain seven restriction sites within a part of the lacZ gene. Insertion of foreign DNA into those sites can be easily detected in E. coli and hybrid plasmids can subsequently be transformed into B. subtilis.