In Vivo and in Vitro Analyses of the AmyR Binding Site of the Aspergillus nidulans agdA Promoter; Requirement of the CGG Direct Repeat for Induction and High Affinity Binding of AmyR

The α-glucosidase gene (agdA) of Aspergillus nidulans has a single CGGN₈CGG type AmyR binding site in its promoter region. The binding site is functional in vivo as a cis-element responsible for induction by starch, and mutational studies indicated that both the CGG triplets are required for high-level induction. A part of AmyR (residues 1-411; AmyR_1-411), which was produced as a MalE fusion protein in E. coli, bound to the CGGN₈CGG site of the agdA promoter. DNA binding profiles to the mutant binding sites that lacked both or either one of the CGG triplets suggested that AmyR_1-411 can bind to a single CGG triplet site with low affinity and that two AmyR molecules cooperatively bind to the CGG direct repeat.     

In vivo footprinting identifies an activating element of the maize Adh2 promoter specific for root and vascular tissues

In vivo footprinting identifies four putative cis elements of Adh2 that interact with protein factors within the DNase I hypersensitive domains of the 5′ flanking region. The power of in vivo footprinting to identify functionally significant sites within a gene promoter was tested by biochemical and transgenic analyses of the putative element at position −160. Biochemical analyses show that proteins isolated from maize cell suspensions will bind to the Adh2 promoter in vitro to generate a footprint at −160 identical to that seen in vivo. The partially purified factor bound to the promoter in vitro can be specifically competed with fragments of DNA containing the element sequence, further demonstrating that a specific protein generates the footprint over that sequence. Transgenic analyses indicate that the −160 element is a functional element of the maize Adh2 promoter that acts as an activator in the meristem and vascular tissue of roots and in the vascular tissue of stems and leaves.     

Highly fluorescent GFPm2+‐based genome integration‐proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages

Study of activity of cloned promoters in slow‐growing Mycobacterium tuberculosis during long‐term growth conditions in vitro or inside macrophages, requires a genome‐integration proficient promoter probe vector, which can be stably maintained even without antibiotics, carrying a substrate‐independent, easily scorable and highly sensitive reporter gene. In order to meet this requirement, we constructed pAKMN2, which contains mycobacterial codon‐optimized gfp_m²⁺ gene, coding for GFP_m²⁺ of highest fluorescence reported till date, mycobacteriophage L5 attP‐int sequence for genome integration, and a multiple cloning site. pAKMN2 showed stable integration and expression of GFP_m²⁺ from M. tuberculosis and M. smegmatis genome. Expression of GFP_m²⁺, driven by the cloned minimal promoters of M. tuberculosis cell division gene, ftsZ (MtftsZ), could be detected in the M. tuberculosis/pAKMN2‐promoter integrants, growing at exponential phase in defined medium in vitro and inside macrophages. Stable expression from genome‐integrated format even without antibiotic, and high sensitivity of detection by flow cytometry and fluorescence imaging, in spite of single copy integration, make pAKMN2 useful for the study of cloned promoters of any mycobacterial species under long‐term in vitro growth or stress conditions, or inside macrophages.     

The int genes of bacteriophages P22 and λ are regulated by different mechanisms

Bacteriophage P22 and λ are related bacteriophages with similar gene organizations. In λ the cII-dependent P_I promoter is responsible for λint gene expression. The only apparent counterpart to P_I in P22 is oriented in the opposite direction, and cannot transcribe the P22 int gene. We show that this promoter, called P_al, is active both in vivo and in vitro, and is dependent upon the P22 cII-like gene, called c1. We have also determined the DNA sequence of a 3.3 kb segment that closes the gap between previously reported sequences to give a continuous sequence between the P22 p_L promoter and the int gene. The newly determined sequence is densely packed with genes from the p_L direction, and the proteins predicted by the sequence show excellent correlation with the proteins mapped by Youderian and Susskind in 1980. However, the sequence contains no apparent genes in the opposite (p_al) direction, and no additional binding motifs for the P22 c1 protein. We conclude that int gene expression in P22 is regulated by a different mechanism than in λ.     

RNA1 is sufficient to mediate plasmid ColE1 incompatibility in vivo

The multicopy plasmid ColE1 specifies a small RNA designated RNA1 that has been implicated in copy number control and incompatibility. We have inserted a 148 base-pair ColE1 DNA fragment containing a promoter-less RNA1 gene into a plasmid vector downstream from the tryptophan promoter of Serratia marcesens. The ColE1 RNA1 produced by this plasmid is not functional in vivo due to the presence of 49 nucleotides appended to the 5′-terminus of the wild-type RNA1 sequence. Deletions of these sequences by Bal3I nuclease in vitro and genetic selection for ColE1 incompatibility function in vivo permitted isolation of a plasmid expressing wild-type ColE1 RNA1 initiated properly from the S. marcesens trp promoter. These experiments demonstrate that RNA1 is sufficient to mediate ColE1 incompatibility in vivo. In addition, several plasmids were isolated that contain altered RNA1 genes. These alterations consist of additions or deletions of sequences at the 5′-terminus of RNA1. Analysis of the ability of these altered RNA1 molecules to express incompatibility in vivo suggests that the 5′-terminal region of RNA1 is crucial for its function.     

5'‐ and 3'‐sequences of satellite tobacco necrosis virus RNA promoting translation in tobacco

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5′ cap and a 3′ poly(A) tail. The STNV trailer contains an autonomous translational enhancer domain (TED) that promotes translation in vitro by more than one order of magnitude when combined with the 5′-terminal 173 nt of STNV RNA. We now show that the responsible sequence within the 5′ region maps to the first 38 nt of the STNV RNA. Mutational analysis indicated that the primary sequence of the STNV 5′ 38 nt and TED is important for translation stimulation in vitro, but did not reveal a role for the complementarity between the two. Translation of chimeric STNV-cat RNAs in tobacco protoplasts showed that TED promotes translation in vivo of RNAs lacking a cap and/or a poly(A) tail. Similar to in vitro, TED-dependent translation in tobacco was stimulated further by the STNV 5′ 38 nt.