Genetic fusions defining trp and lac operon regulatory elements

Two procedures for easily isolating deletions that fuse the trp and lac operons are described. Using these procedures, a large number of fusion deletions have been isolated. The lac ends of these deletions extend varying distances into the lacI gene and the lac promoter-operator region. Therefore, contrary to a previous report, there does not appear to be a messenger-termination signal at the C-terminal end of the lacI gene.The trp ends of fusion deletions do not have to extend into the trp structural genes to effect fusion, suggesting that mRNA synthesis initiated at the trp promoter proceeds some distance beyond the trp structural genes before a messenger termination signal is reached. Deletions that extend a short distance into the C-terminus of trpA, the last gene in the trp operon, do not completely abolish activity of the trpA product.The procedures described for isolating fusions of the trp and lac operons can be generalized to other systems.     

Isolation and functional characterization of two novel seed-specific promoters from sunflower (Helianthus annuus L.)

The promoter region of two sunflower (Helianthus annuus L. HA89 genotype) seed specifically expressed genes, coding for an oleate desaturase (HaFAD2-1) and a lipid transfer protein (HaAP10), were cloned and in silico characterized. The isolated fragments are 867 and 964 bp long, respectively, and contain several seed-specific motifs, such as AACA motif, ACGT element, E-Boxes, SEF binding sites and GCN4 motif. Functional analysis of these promoters in transgenic Arabidopsis plants was investigated after fusing them with the β-glucuronidase (GUS) reporter gene. None of the promoters triggered GUS activity in any vegetative tissue, with the exception of early seedling cotyledons. HaFAD2-1 and HaAP10 promoters were tested along seed development from globular stage to mature seeds. GUS staining was restricted to embryonic tissue and quantitative fluorometric assays showed high activity values at the later stages of development. In this work we demonstrate that HaFAD2-1 promoter is as strong as 35S promoter even though it is a tissue-specific promoter and its activity derived just from the embryo, thus confirming that it can be considered a strong highly specific seed promoter useful for biotechnology applications.     

Isolation and characterization of Lactococcus lactis subsp. lactis promoters.

DNA fragments with promoter activity were isolated from the chromosome of Lactococcus lactis subsp. lactis. For the isolation, a promoter probe vector based on the cat gene was constructed, which allowed direct selection with chloramphenicol in Bacillus subtilis and L. lactis. Four of the putative promoters (P1, P2, P10, and P21) were analyzed further by sequencing, mapping of the 5' end of the mRNA, Northern (RNA blot) hybridization, and chloramphenicol acetyltransferase activity measurements. From these fragments, -10 and -35 regions resembling the consensus Escherichia coli sigma 70 and B. subtilis sigma 43 promoters were identified. Another set of promoters, together with a signal sequence, were also isolated from the same organism. These fragments promoted secretion of TEM beta-lactamase from L. lactis. When the two sets of promoters were compared, it was found that the ones isolated with the cat vector were more efficient (produced more mRNA). By changing the promoter part of the promoter-signal sequence fragment giving the best TEM beta-lactamase secretion into a more efficient one (P2), a 10-fold increase in enzyme production was obtained.     

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.     

Isolation and characterization of root-specific phosphate transporter promoters from Medicago trunatula

Promoters of phosphate transporter genes MtPT1 and MtPT2 of Medicago truncatula were isolated by utilizing the gene-space sequence information and by screening of a genomic library, respectively. Two reporter genes, beta-glucuronidase (GUS) and green fluorescent protein (GFP) were placed under the control of the MtPT1 and MtPT2 promoters. These chimeric transgenes were introduced into Arabidopsis thaliana and transgenic roots of M. truncatula, and expression patterns of the reporter genes were assayed in plants grown under different phosphate (Pi) concentrations. The expression of GUS and GFP was only observed in root tissues, and the levels of expression decreased with increasing concentrations of Pi. GUS activities in roots of transgenic plants decreased 10-fold when the plants were transferred from 10 microM to 2 mM Pi conditions, however, when the plants were transferred back to 10 microM Pi conditions, GUS expression reversed back to the original level. The two promoters lead to different expression patterns inside root tissues. The MtPT1 promoter leads to preferential expression in root epidermal and cortex cells, while MtPT2 promoter results in strong expression in the vascular cylinder in the center of roots. Promoter deletion analyses revealed possible sequences involved in root specificity and Pi responsiveness. The promoters are valuable tools for defined engineering of plants, particularly for root-specific expression of transgenes.     

Isolation and characterization of Lactococcus lactis subsp. lactis promoters.

DNA fragments with promoter activity were isolated from the chromosome of Lactococcus lactis subsp. lactis. For the isolation, a promoter probe vector based on the cat gene was constructed, which allowed direct selection with chloramphenicol in Bacillus subtilis and L. lactis. Four of the putative promoters (P1, P2, P10, and P21) were analyzed further by sequencing, mapping of the 5' end of the mRNA, Northern (RNA blot) hybridization, and chloramphenicol acetyltransferase activity measurements. From these fragments, -10 and -35 regions resembling the consensus Escherichia coli sigma 70 and B. subtilis sigma 43 promoters were identified. Another set of promoters, together with a signal sequence, were also isolated from the same organism. These fragments promoted secretion of TEM beta-lactamase from L. lactis. When the two sets of promoters were compared, it was found that the ones isolated with the cat vector were more efficient (produced more mRNA). By changing the promoter part of the promoter-signal sequence fragment giving the best TEM beta-lactamase secretion into a more efficient one (P2), a 10-fold increase in enzyme production was obtained.     

Improved plasmid vectors for the isolation of translational lac gene fusions

The beta-galactosidase fusion vector pMC1403 has been modified to include the unique cloning sites EcoRI, SmaI, BamHI, SalI, AccI, PstI and HindIII. The new vectors (pNM480, pNM481 and pNM482) allow the fusion of genes to beta-galactosidase in all three translational reading frames, and exhibit an increased sensitivity of promoter detection due to a higher copy number.     

Characterization of fusions between the lac operon and the ilv gene cluster in Escherichia coli: ilvC-lac fusions.

By means of the general procedure of Casadaban (J. Mol. Biol. 104: 541-556, 1976), the lac genes carried on a lambda-Mu-1 hybrid phage were inserted into a temperature-inducible Mu-1 prophage that had earlier been inserted into a site near the beginning of the ilvC gene of Escherichia coli strain K-12. Selection of temperature-resistant derivatives of the lysogen resulted in a fusion of the lac genes to a region of deoxyribonucleic acid that is transcribed under the control of the ilvC regulatory elements. A strain bearing the fusion was shown to be inducible for beta-galactosidase by acetohydroxybutyrate, a natural inducer of acetohydroxy acid isomeroreductase. Induction of the lysogen by mitomycin C led to the isolation of a plaque-forming lambda derivative carrying this ilvC-lac fusion.     

Construction of lac fusions to the inducible arginine-and lysine decarboxylase genes of Escherichia coli K12

The induction of several amino acid decarboxylases under anaerobic conditions at low pH has been known for many years, but the mechanism associated with this type of regulation has not been elucidated. To study the regulation of the biodegradative arginine and lysine decarboxylases of Escherichia coli K12, Mudlac fusions to these genes were isolated. Mudlac fusion strains deficient for lysine decarboxylase or arginine decarboxylase were identified using decarboxylase indicator media and analysed for their regulation of beta-galactosidase expression. The position of the Mudlac fusion in lysine decarboxylase-deficient strains has been mapped to the cadA gene at 93.7 minutes, while the Mudlac fusions exhibiting a deficiency in the inducible arginine decarboxylase have been mapped to 93.4 minutes.