Cis DNA Research Articles

Transcriptional regulation of the mouse angiotensin II type 2 receptor gene.

The promoter region of the mouse angiotensin II type 2 receptor gene was cloned, and the nucleotide sequences were determined. A computer homology search for a 1.5-kb promoter region showed that there are several consensus cis DNA elements such as C/EBP, NF-IL6, and AP-1 in this region. Primer extension experiments showed that there are two transcription initiation sites 16 bp apart in the mouse type 2 receptor gene. Deletion mutants of this 1.5-kb segment were prepared and fused to a luciferase reporter gene. These type 2 receptor promoter-luciferase constructs were introduced into PC12W cells, which are from a pheochromocytoma cell line expressing the type 2 receptor, and luciferase activity was measured. It showed that a DNA segment between nucleotides -1497 and -874 suppresses the promoter activity of the type 2 receptor gene and that a DNA segment between nucleotides -47 and +56 is important for the basal promoter activity of the type 2 receptor gene. This proximal segment showed very weak promoter activity when introduced into vascular smooth muscle cells. Gel mobility shift assay with nuclear extracts from PC12W cells showed the presence of three DNA binding proteins that bound to a DNA probe between nucleotides -47 and +8. One DNA binding protein was only very weakly expressed in nuclear extracts from vascular smooth muscle cells, which do not express the type 2 receptor. Two other DNA binding proteins were not observed in nuclear extracts from vascular smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Lambda integrase cleaves DNA in cis.

In the Int family of site-specific recombinases, DNA cleavage is accomplished by nucleophilic attack on the activated scissile phosphodiester bond by a specific tyrosine residue. It has been proposed that this tyrosine is contributed by a protomer bound to a site other than the one being cleaved ('trans' cleavage). To test this hypothesis, the difference in DNA binding specificity between closely related integrases (Ints) from phages lambda and HK022 was exploited to direct wild type Ints and cleavage- or activation-defective mutants to particular sites on bispecific substrates. Analysis of Int cleavage at individual sites strongly indicates that DNA cleavage is catalyzed by the Int bound to the cleaved site ('cis' cleavage). This conclusion contrasts with those from previous experiments with two members of the Int family, FLP and lambda Int, that supported the hypothesis of trans cleavage. We suggest explanations for this difference and discuss the implications of the surprising finding that Int-family recombinases appear capable of both cis and trans mechanisms of DNA cleavage.

A 70- to 80-kDa Glial Cell Protein Interacts with the AGGGAAGGGA Domain of the JC Virus Early Promoter Only in the Presence of the Neighboring cis DNA Elements

The neurotropic virus JC (JCV) causes the demyelinating disease progressive multifocal leukoencephalopathy. JCV is transcriptionally regulated by both positive and negative cis elements. Previous studies have shown that the AGGGAAGGGA sequence, in isolation, interacts with a 53- to 56-kDa cellular protein and acts as a silencer for the late promoter. We have reexamined the role of the AGGGAAGGGA element in the glial cell-specific transcriptional regulation of JCV by studying it in a longer natural context of elements of the JCV early promoter. Transcriptional analysis in C6 gliel cells of selectively mutated AGGGAAGGGA domains within the nt 98-153 region of the JCV early promoter revealed that both GGG regions in the AGGGAAGGGA sequence are required for glial cell-specific transcription. The interactions of cellular proteins with the AGGGAAGGGA domain were also found to be dependent upon the presence or absence of neighboring cis elements The TATA region was found to facilitate the interaction of specific proteins with the AGGGAAGGGA domain. Domain-specific photocrosslinking studies revealed the binding of a glial cell protein of approximately 70-80 kDa to the AGGGAAGGGA domain only when it was flanked by its neighboring cis elements, the TATA and NF-1 domains of the JCV promoter.

Cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication

Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA.

T-cell receptor gamma delta and gamma transgenic mice suggest a role of a gamma gene silencer in the generation of alpha beta T cells.

A T lymphocyte expresses on its surface one of two types of antigen receptor, T-cell receptor alpha beta or T-cell receptor gamma delta, encoded by a pair of somatically rearranged alpha and beta or gamma and delta genes. It has been suggested that alpha beta T cells are generated only from precursor T cells that failed to rearrange gamma and delta genes in a functional form. However, we found that transgenic mice constructed with functionally rearranged gamma and delta genes produce a normal number of alpha beta T cells. The transgene gamma present in these alpha beta T cells is repressed apparently through an associated cis DNA element (silencer). We propose that some T-cell precursors are committed to generate alpha beta T cells independent of the rearrangement status of their gamma gene and that this commitment involves activation of a factor(s) that interacts with the gamma gene-associated silencer.

Construction, expression, and localization of a CycA::PhoA fusion protein in Rhodobacter sphaeroides and Escherichia coli

We demonstrated the utility of Escherichia coli alkaline phosphatase, encoded by phoA, as a reporter molecule for genetic fusions in Rhodobacter sphaeroides. A portion of the R. sphaeroides cycA gene was fused to phoA, yielding a fusion protein comprising the putative signal sequence and first 10 amino acids of the cytochrome c2 apoprotein joined to the sixth amino acid of alkaline phosphatase. The fusion protein was efficiently transported to the periplasm of R. sphaeroides as determined by enzyme activity, Western immunoblot analysis, and immunogold electron microscopy. We also documented the ability of an R. sphaeroides mutant, RS104, with gross defects in photosynthetic membrane morphology to efficiently recognize and translocate the fusion protein to the periplasmic compartment. The inclusion of 500 base pairs of R. sphaeroides DNA in cis to the cycA structural gene resulted in a 2.5-fold increase in alkaline phosphatase activity in photosynthetically grown cells compared with the activity in aerobically grown cells, demonstrating that the fusion protein is regulated in a manner similar to that of cytochrome c2 regulation. We also constructed two pUC19-based plasmids suitable for the construction of translational fusions to phoA. In these plasmids, translational fusions of phoA to the gene under consideration can be made in all three reading frames, thus facilitating construction and expression of fusion protein systems utilizing phoA.