No abstract

An improved vector system has been developed for the in vitro construction of transcriptional fusions to lacZ. The principal feature is an RNaseIII cleavage site inserted between the polylinker cloning site and the promoterless lacZ gene. When these vectors are used to construct transcriptional fusions, the subsequent cleavage of the hybrid mRNA at the RNaseIH site generates an unchanging 5' end for the lacZ mRNA. In contrast to earlier vectors, this feature helps to ensure independent translation of the lacZ mRNA and, thus, the level of ,l-galactosidase produced should accurately reflect the frequency of transcription of the upstream DNA sequences. Additional modifications of the vectors include removal of a weak transcriptional terminator between the cloning site and lacZ, insertion of a terminator downstream of lac, and alteration of restriction endonuclease cleavage sites to facilitate the in vitro construction of fusions. Both multicopy plasmid (pTL61T) and single-copy lambda (ATL61) vectors have been assembled. These vectors should be generally useful in scanning for transcriptional regulatory signals.Gene fusions have become important tools for the analysis of gene regulation in both procaryotic and eucaryotic systems. In general, the attachment of the regulatory sites of a given gene upstream of a reporter gene that encodes an easily assayed enzyme facilitates the analysis of expression of the affixed gene. The reporter gene may be used in either transcriptional (operon) fusions, where it retains its own translational start site but is dependent on the attached DNA for transcription, or in translational (protein) fusions, where both its transcription and translation are dependent on signals in the attached upstream DNA. The history and a wide variety of specific applications of gene fusions have been reviewed by Silhavy and Beckwith (45).Although a variety of reporter genes have been used in the construction of gene fusions, the most common is lacZ of Escherichia coli. The product, 3-galactosidase, is stable in many cellular backgrounds and can be precisely quantitated by a very simple and sensitive colorimetric assay. Moreover, the construction of fusions to lacZ extends all the experimental convenience of the lac genetic system to any given gene. That is, by using the available lac technology, one can readily screen and select for altered levels of f-galactosidase produced from the gene fusion. This greatly facilitates the regulatory analysis of the upstream attached gene.Numerous vector systems have been described for the construction of gene fusions to lacZ (45). Both multicopy (plasmid) and single-copy (bacteriophage) vectors have been used to construct gene fusions by either in vivo or in vitro manipulations. We previously described a vector system for the in vitro construction of transcriptional fusions to lacZ which had some advantages over the then currently available vectors (26). Both plasmid (pTL25) and phage (XTL25) versions were assembled, but we have used primarily the single-copy vector...

Protein kinase CK2 (formerly casein kinase II) exhibits elevated expression in a variety of cancers, induces lymphocyte transformation in transgenic mice, and collaborates with Ha-Ras in fibroblast transformation. To systematically examine the cellular functions of CK2, human osteosarcoma U2-OS cells constitutively expressing a tetracycline-regulated transactivator were stably transfected with a bidirectional plasmid encoding either catalytic isoform of CK2 (i.e. CK2␣ or CK2␣) together with the regulatory CK2␤ subunit in order to increase the cellular levels of either CK2 isoform. To interfere with either CK2 isoform, cells were also transfected with kinase-inactive CK2␣ or CK2␣ (i.e. GK2␣ (K68M) or CK2␣(K69M)) together with CK2␤. In these cells, removal of tetracycline from the growth medium stimulated coordinate expression of catalytic and regulatory CK2 subunits. Increased expression of active forms of CK2␣ or CK2␣ resulted in modest decreases in cell proliferation, suggesting that optimal levels of CK2 are required for optimal proliferation. By comparison, the effects of induced expression of kinase-inactive CK2␣ differed significantly from the effects of induced expression of kinase-inactive CK2␣. Of particular interest is the dramatic attenuation of proliferation that is observed following induction of CK2␣(K69M), but not following induction of CK2␣(K68M). These results provide evidence for functional specialization of CK2 isoforms in mammalian cells. Moreover, cell lines exhibiting regulatable expression of CK2 will facilitate efforts to systematically elucidate its cellular functions. Protein kinase CK21 (formerly casein kinase II) is a ubiquitously distributed and highly conserved protein serine/threonine kinase that is essential for viability in eukaryotes (1, 2). Although its precise functions remain poorly understood, there is mounting evidence to suggest that CK2 plays an important role in the control of cell proliferation and transformation (3-6). Alterations in the expression of CK2 have been observed in a variety of tumor or leukemic cells (7-13), and in the lymphocytes of cattle that develop T cell lymphomas following infection with the parasite Theileria parva (14). Furthermore, the targeted overexpression of CK2␣ in the T cells of transgenic mice results in the development of lymphomas (15). By crossing different lines of transgenic mice, there is evidence for collaboration between the dysregulated expression of CK2␣ and the c-Myc and Tal-1 oncogenes in lymphoma development (16). Accelerated lymphomagenesis is also observed when the mice that overexpress CK2 in T cells are crossed with mice deficient in the functional expression of p53 (17). There is also evidence that CK2 contributes to the transformation of primary and established fibroblasts. Overexpression of either catalytic isoform of CK2 (i.e. CK2␣ or CK2␣Ј) exhibited cooperativity with Ha-Ras in the transformation of rat embryo fibroblasts and Balb/c 3T3 cells (18). All of these observations suggest that the increased expression of CK2...

Numerous experiments have indicated that the synthesis of RNA polymerase (beta beta' alpha 2 sigma 70) in Escherichia coli is autogenously regulated. In the present study, we have examined expression of the rpoB and rpoC genes which encode the beta and beta' subunits of RNA polymerase. These genes are the distal cistrons of the rplKAJLrpoBC ribosomal protein-RNA polymerase transcription unit. Both transcriptional (operon) and translational (gene) fusions of either rpoB or rpoC to the lacZ reporter were used to monitor their in vivo expression by inserting single copies of these fusions into the bacterial chromosome on integration-proficient lambda vectors. In order to examine the expression of the rpoBC genes in response to varied intracellular concentrations of the RNA polymerase subunits, a series of expression plasmids carrying the rpoB, rpoC, rpoA (alpha) and rpoD (sigma 70) genes in different combinations were then introduced into these cells. Elevated concentrations of either beta or beta' were found to repress the expression of both rpoB and rpoC at the translational level. However, the simultaneous increase in the concentration of all the subunits that comprise holoenzyme repressed the transcription of rpoBC. To determine the site of this repression, additional operon fusions were utilized which placed lacZ downstream of each of the transcriptional regulatory sites of this gene cluster, including two promoters, rplKp and rplJp, and a transcriptional attenuator in the rplL-rpoB intercistronic region. Expression from these fusions showed that transcriptional repression is achieved primarily by reducing initiation at both upstream promoters, coupled with a small increase in termination at the attenuator.

A protocol has been developed that is capable of saturating regions hundreds of basepairs in length with linker scanning mutations. The efficacy of this method stems from the design of the linker scanning mutagenesis (LSM) cassette which is composed of a selectable marker flanked by two oligonucleotides, each of which contains a recognition site for a different restriction endonuclease. The cleavage site for one endonuclease is within its recognition site, while the second endonuclease cleaves in the target DNA beyond the end of the cassette. Digestion with these endonucleases and subsequent ligation results in the replacement of 12 bp of the original target sequence with 12 bp of the linker scanning oligonucleotide. We have used this protocol to mutagenize a span of approximately 400 bp surrounding the start site of the gene for the beta subunit (rpoB) of Escherichia coli RNA polymerase. The translation of the beta mRNA has been shown previously to be regulated by the intracellular concentration of either beta or beta'. Analysis of the linker scanning mutations indicates that sequences extending a considerable distance both upstream and downstream of the start site are required for normal translation. Also a site that appears to be involved in translational repression by excess beta' has been identified.