Dependence on reporter gene of apparent activity in gene fusions of a Streptomyces griseus streptomycin biosynthesis promoter

1995 ◽  
Vol 41 (4-5) ◽  
pp. 407-417 ◽  
Author(s):  
Helen K. Lindley ◽  
V. Jayne Deeble ◽  
Ursula Peschke ◽  
Mary O'Neill ◽  
Simon Baumberg ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Copy Number ◽  
Streptomyces Griseus ◽  
Gene Fusions ◽  
Leader Region ◽  
Short Fragment ◽  
Copy Number Plasmid ◽  
Streptomycin Biosynthesis ◽  
Low Copy Number

The adjacent genes strR–strA–strB1 lie within the large cluster of genes of streptomycin biosynthesis and resistance in Streptomyces griseus. sirR encodes a pathway-specific activator StrR, suggested by previous work to be either an antiterminator or a conventional activator, binding to its DNA target via a helix-turn-helix motif. strB1 is transcribed in an StrR-dependent fashion from a promoter (PstrB1) that lies downstream from strA; between PstrB1 and sirB1 there is a 300-bp leader region containing numerous inverted repeats that could represent modulatable transcription termination sites. Hybrid plasmids were constructed in vitro with transcriptional fusions in which fragments containing PstrB1 and either the entire leader region ("long" fragments) or a small part of it (the "short" fragment) were cloned upstream of (i) aph as reporter gene, in a high copy number plasmid background, or (ii) xylE as reporter gene, in a low copy number plasmid background. The short fragment directed high levels of APH (aminoglycoside 3′-phosphotransferase) whether StrR was present or not, while the long fragments did not do so in the absence of StrR; one long fragment directed high levels in wild-type S. griseus, in which StrR would be present. Insertion of an extraneous fragment into PstrB1 in the short fragment construct led to loss of APH activity, demonstrating that no adventitious promoter had been formed in the short construct. In vitro deletion of part of the leader region in a long fragment construct led to high APH expression with or without StrR present. Although these results are consistent with the target of StrR being within the leader region, and thus with an antiterminator role, it was found that both long and short fragments in the low copy number background failed to direct high expression of catechol oxygenase (the product of xylE) unless strR was also present on a compatible plasmid. Transfer of PstrB1-xylE fragments to the high copy number vector did not increase catechol oxygenase expression. We interpret these results in terms of an effect, in the hybrid constructs, of one of the reporter genes on promoter function, possibly by affecting local DNA topology.Key words: gene fusions, reporter genes, Streptomyces, streptomycin, regulation of secondary metabolism.

scholarly journals Synergistic activation of ADH2 expression is sensitive to upstream activation sequence 2 (UAS2) orientation, copy number and UAS1-UAS2 helical phasing.

1995 ◽  
Vol 15 (6) ◽  
pp. 3442-3449 ◽  
Author(s):  
M S Donoviel ◽  
N Kacherovsky ◽  
E T Young
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Reporter Gene ◽  
Copy Number ◽  
Glucose Repression ◽  
Regulatory Protein ◽  
Regulatory Region ◽  
Upstream Regulatory Region ◽  
Specific Sequence

The alcohol dehydrogenase 2 (ADH2) gene of Saccharomyces cerevisiae is under stringent glucose repression. Two cis-acting upstream activation sequences (UAS) that function synergistically in the derepression of ADH2 gene expression have been identified. UAS1 is the binding site for the transcriptional regulator Adr1p. UAS2 has been shown to be important for ADH2 expression and confers glucose-regulated, ADR1-independent activity to a heterologous reporter gene. An analysis of point mutations within UAS2, in the context of the entire ADH2 upstream regulatory region, showed that the specific sequence of UAS2 is important for efficient derepression of ADH2, as would be expected if UAS2 were the binding site for a transcriptional regulatory protein. In the context of the ADH2 upstream regulatory region, including UAS1, working in concert with the ADH2 basal promoter elements, UAS2-dependent gene activation was dependent on orientation, copy number, and helix phase. Multimerization of UAS2, or its presence in reversed orientation, resulted in a decrease in ADH2 expression. In contrast, UAS2-dependent expression of a reporter gene containing the ADH2 basal promoter and coding sequence was enhanced by multimerization of UAS2 and was independent of UAS2 orientation. The reduced expression caused by multimerization of UAS2 in the native promoter was observed only in the presence of ADR1. Inhibition of UAS2-dependent gene expression by Adr1p was also observed with a UAS2-dependent ADH2 reporter gene. This inhibition increased with ADR1 copy number and required the DNA-binding activity of Adr1p. Specific but low-affinity binding of Adr1p to UAS2 in vitro was demonstrated, suggesting that the inhibition of UAS2-dependent gene expression observed in vivo could be a direct effect due to Adr1p binding to UAS2.

scholarly journals Targeted Isolation of a Designated Region of the Bacillus subtilis Genome by Recombinational Transfer

2004 ◽  
Vol 70 (4) ◽  
pp. 2508-2513 ◽  
Author(s):  
Satoshi Tomita ◽  
Kenji Tsuge ◽  
Yo Kikuchi ◽  
Mitsuhiro Itaya
Keyword(s):  
Bacillus Subtilis ◽  
Copy Number ◽  
Positional Cloning ◽  
Deletion Mutant ◽  
Essential Gene ◽  
Transfer System ◽  
Copy Number Plasmid ◽  
Low Copy Number ◽  
Subtilis Genome

ABSTRACT A method for positional cloning of the Bacillus subtilis genome was developed. The method requires a set of two small DNA fragments that flank the region to be copied. A 38-kb segment that carries genes ppsABCDE encoding five enzymes for antibiotic plipastatin synthesis and another genome locus as large as 100 kb including one essential gene were examined for positional cloning. The positional cloning vector for ppsABCDE was constructed using a B. subtilis low-copy-number plasmid that faithfully copied the precise length of the 38-kb DNA in vivo via the recombinational transfer system of this bacterium. Structure of the copied DNA was confirmed by restriction enzyme analyses. Furthermore, the unaltered structure of the 38-kb DNA was demonstrated by complementation of a ppsABCDE deletion mutant.

scholarly journals A vector for studying plasmid stability functions in Streptomyces

1988 ◽  
Vol 51 (1) ◽  
pp. 71-74 ◽  
Author(s):  
Kevin Kendall ◽  
John Cullum
Keyword(s):  
Copy Number ◽  
Stability Region ◽  
Selectable Marker ◽  
Plasmid Stability ◽  
Stability Function ◽  
Melanin Production ◽  
Stability Functions ◽  
Tyrosinase Gene ◽  
Copy Number Plasmid ◽  
Low Copy Number

SummaryWe constructed a cloning vector (pMT603) based on the low copy number plasmid SCP2*. pMT6O3 is unstable because it lacks the SCP2* stability region and carries the selectable marker thiostrepton-resistance and a tyrosinase gene which results in melanin production. This allows easy testing of plasmid stability and we demonstrated its usefulness by cloning a plasmid stability function.

scholarly journals A Low-Copy-Number Plasmid for Retrieval of Toxic Genes from BACs and Generation of Conditional Targeting Constructs

2012 ◽  
Vol 54 (2) ◽  
pp. 504-514 ◽  
Author(s):  
Giyoun Na ◽  
Andrew Wolfe ◽  
CheMyong Ko ◽  
Hyesook Youn ◽  
Young-Min Lee ◽  
...  
Keyword(s):  
Copy Number ◽  
Copy Number Plasmid ◽  
Low Copy Number

scholarly journals Replication of a low-copy-number plasmid by a plasmid DNA-membrane complex extracted from minicells of Escherichia coli.

1982 ◽  
Vol 150 (3) ◽  
pp. 1234-1243 ◽  
Author(s):  
W Firshein ◽  
P Strumph ◽  
P Benjamin ◽  
K Burnstein ◽  
J Kornacki
Keyword(s):  
Escherichia Coli ◽  
Plasmid Dna ◽  
Copy Number ◽  
Membrane Complex ◽  
Copy Number Plasmid ◽  
Low Copy Number

scholarly journals The Bacteriophage P1 HumD Protein Is a Functional Homolog of the Prokaryotic UmuD′-Like Proteins and Facilitates SOS Mutagenesis in Escherichia coli

1999 ◽  
Vol 181 (22) ◽  
pp. 7005-7013 ◽  
Author(s):  
Mary P. McLenigan ◽  
Olga I. Kulaeva ◽  
Don G. Ennis ◽  
Arthur S. Levine ◽  
Roger Woodgate
Keyword(s):  
Escherichia Coli ◽  
Copy Number ◽  
Dna Lesions ◽  
Broad Host Range ◽  
Bacteriophage P1 ◽  
Copy Number Plasmid ◽  
Functional Homolog ◽  
Vitro Analysis

ABSTRACT The Escherichia coli umuD and umuC genes comprise an operon and encode proteins that are involved in the mutagenic bypass of normally replication-inhibiting DNA lesions. UmuD is, however, unable to function in this process until it undergoes a RecA-mediated cleavage reaction to generate UmuD′. Many homologs ofumuDC have now been identified. Most are located on bacterial chromosomes or on broad-host-range R plasmids. One such putative homolog, humD (homolog of umuD) is, however, found on the bacteriophage P1 genome. Interestingly,humD differs from other umuD homologs in that it encodes a protein similar in size to the posttranslationally generated UmuD′ protein and not UmuD, nor is it in an operon with a cognate umuC partner. To determine if HumD is, in fact, a bona fide homolog of the prokaryotic UmuD′-like mutagenesis proteins, we have analyzed the ability of HumD to complement UmuD′ functions in vivo as well as examined HumD’s physical properties in vitro. When expressed from a high-copy-number plasmid, HumD restored cellular mutagenesis and increased UV survival to normally nonmutablerecA430 lexA(Def) and UV-sensitive ΔumuDC recA718 lexA(Def) strains, respectively. Complementing activity was reduced when HumD was expressed from a low-copy-number plasmid, but this observation is explained by immunoanalysis which indicates that HumD is normally poorly expressed in vivo. In vitro analysis revealed that like UmuD′, HumD forms a stable dimer in solution and is able to interact with E. coli UmuC and RecA nucleoprotein filaments. We conclude, therefore, that bacteriophage P1 HumD is a functional homolog of the UmuD′-like proteins, and we speculate as to the reasons why P1 might require the activity of such a protein in vivo.

Sign in / Sign up

Export Citation Format

Share Document