Peptide nucleic acids (PNAs) are polyamide oligomers that can strand invade duplex DNA, causing displacement of one DNA strand and formation of a D-loop. Binding of either a T10 PNA or a mixed sequence 15-mer PNA to the transcribed strand of a G-free transcription cassette caused 90 to 100 percent site-specific termination of pol II transcription elongation. When a T10 PNA was bound on the nontranscribed strand, site-specific inhibition never exceeded 50 percent. Binding of PNAs to RNA resulted in site-specific termination of both reverse transcription and in vitro translation, precisely at the position of the PNA.RNA heteroduplex. Nuclear microinjection of cells constitutively expressing SV40 large T antigen (T Ag) with either a 15-mer or 20-mer PNA targeted to the T Ag messenger RNA suppressed T Ag expression. This effect was specific in that there was no reduction in beta-galactosidase expression from a coinjected expression vector and no inhibition of T Ag expression after microinjection of a 10-mer PNA.

Progression through the somatic cell cycle requires the temporal regulation of cyclin gene expression and cyclin protein turnover. One of the best-characterized examples of this regulation is seen for the B-type cyclins. These cyclins and their catalytic component, cdc2, have been shown to mediate both the entry into and maintenance of mitosis. The cyclin B1 gene has been shown to be expressed between the late S and G 2 phases of the cell cycle, while the protein is degraded specifically at interphase via ubiquitination. To understand the molecular basis for transcriptional regulation of the cyclin B1 gene, we cloned the human cyclin B1 gene promoter region. Using a chloramphenicol acetyltransferase reporter system and both stable and transient assays, we have shown that the cyclin B1 gene promoter (extending to ؊3800 bp relative to the cap site) can confer G 2 -enhanced promoter activity. Further analysis revealed that an upstream stimulatory factor (USF)-binding site and its cognate transcription factor(s) are critical for expression from the cyclin B1 promoter in cycling HeLa cells. Interestingly, USF DNA-binding activity appears to be regulated in a G 2 -specific fashion, supporting the idea that USF may play some role in cyclin B1 gene activation. These studies suggest an important link between USF and the cyclin B1 gene, which in part explains how maturation promoting factor complex formation is regulated.Cyclins are a family of related proteins which are present at specific stages of the somatic cell cycle (58). They function as regulatory subunits for cyclin-dependent kinases (cdks), which phosphorylate key substrates that mediate cell cycle transit (37,43,56,58). Catalytic activation of the cdks requires sufficient accumulation of cyclin protein at particular stages of the cell cycle (41-43, 56). Dysregulation of cyclin gene expression through overexpression and/or unscheduled cdk activity results in inappropriate entry into the S or M phase and may be characteristic of some human cancer cells (7,20,26,30,31,38,45,53,60). The best-characterized cyclin-cdk complex is maturation-promoting factor, which consists of a B-type cyclin and cdc2 kinase (13,17,18,33,41,56). The B-type cyclins (B1, B2, and B3), as well as cyclin A, have been implicated in control of the G 2 /M transition (13,16,37,38,41,42,56,58). The interaction between B-type cyclins and cdc2 during the G 2 cell cycle phase is necessary for cdc2 kinase activation, and the resultant phosphorylations mediate structural changes crucial for the G 2 /M transition (12,16,37,43). Activation of the cdc2 kinase does not occur until sufficient cyclin B protein has been synthesized (56), whereas proteolysis of cyclin B via ubiquitination at the end of mitosis is critical for entry into interphase (19). The accumulation of cyclin B protein, as with many other cyclins, is correlated with nascent-gene expression. Cyclin B mRNA can be detected in late S phase, peaks in late G 2 phase, and cannot be detected in M, G 1 , or early S (41). Models that explain the ...

Type 5 adenovirus mutants that differentially express ElA 13S, 12S, or 9S mRNAs were constructed to study the role of their gene products in transformation. H5dl520 expresses the 243-amino-acid (AA) protein encoded in the 12S mRNA but not the 13S mRNA-encoded 289-AA protein. This mutant transformed both cloned rat embryo fibroblast (CREF) cells and baby rat kidney (BRK) cells at a frequency 40-100 times greater than did wild-type viruses. In addition, all of the foci produced were fibroblastic and grew very slowly at 320C. In contrast, H5dl21, which was mutated so that only the 54-AA protein encoded by the 9S mRNA was synthesized, did not transform either cell type. DNA transfection studies with plasmids from which these mutants were constructed demonstrated that the differences in transformation frequencies were not as marked. The plasmid pDl/D2, which directs the synthesis of the 54-AA protein only, was found to transform baby rat kidney cells at low frequency, provided the gene was linked to a fragment from the simian virus 40 genome containing the transcriptional enhancer element.The early region genes ElA and E1B, located at the lefthand end of the genome, have been shown to be essential for complete morphological transformation of rodent cells by human adenoviruses (Ad) (1-4). However, transfections of baby rat kidney (BRK) cells (5) or an established cloned rat embryo fibroblast (CREF) cell line (6) with various fragments of the Ad type 5 (AdS) or type 12 (Adl2) genome containing only the ElA sequences produce partial or incomplete morphological transformation, whereas DNA fragments containing only the E1B genes do not transform (7).The exact roles of the ElA genes in transformation are not well understood, but they do provide an establishment function that is concerned with the immortalization of primary cells (2, 8). The ElA sequences encode three different but related products. During early times of productive infection, the ElA primary transcript is differentially spliced to give 12S and 13S messages that differ in the extent of internal sequences removed by RNA splicing (9-12). The translational reading frames of both messages are identical, and, thus, the proteins encoded by each [a 289-amino-acid (AA) protein by the 13S mRNA and the 243-AA protein by the 12S mRNA] differ only by an additional 46 AA encoded by the sequences unique to the 13S mRNA. At late times of infection, a third message, 9S in size, is synthesized. The predicted polypeptide encoded by this message is 54 AA in size and has the same amino-terminal sequence before the splice as in the protein products of the 12S and 13S mRNAs (13). A wellcharacterized function of the ElA region in the productive infection of cells is to produce a product that hastens the transcription of the other early viral genes (4, [14][15][16] (3,5,18,20); some are transformation defective, whereas, others are cold sensitive for the initiation and maintenance of the transformed phenotype. From these results it was deduced that the 289-AA protein was di...

Polyamide oligomers, termed peptide nucleic acids (PNAs), bind with high affinity to both DNA and RNA and offer both antisense and antigene approaches for regulating gene expression. When a PNA binds to a complementary sequence in a double-stranded DNA, one strand of the duplex is displaced, and a stable D-loop is formed. Unlike oligodeoxynucleotides for which binding polarity is determined by the deoxyribose sugar, the unrestrained polyamide backbone ofthe PNA could permit binding to a DNA target in an orientationindependent manner. We now provide evidence that PNAs can, in fact, bind to their complementary sequence in DNA independent of the DNA-strand polarity-that is, a PNA binds to DNA in both "parallel" and "antiparallel" fashion. With a mixed-sequence 15-mer PNA, kinetic studies of PNADNA interactions revealed that D-loop formation was rapid and the complex was stable for several hours. However, when measured either by gel-mobility-shift analysis or RNA polymerase lI-elongation termination, D-loop formation was salt dependent, but PNA-strand dissociation was not salt dependent. We observed that D-loop-containing DNA fragments had anomalous gel mobilities that varied as a function of the position of the D-loop relative to the DNA termini. On the basis of permutation analysis, the decreased mobility of the PNA-DNA complex was attributed to a bend in the DNA at or near the D-loop.The inhibition of gene expression mediated by oligodeoxynucleotides is a powerful research tool that has been exploited to study the function of specific gene products in complex biological pathways (for reviews, see refs. 1 and 2). Although technically challenging, inhibiting gene expression at the level of transcriptional initiation and/or RNA polymerase elongation (antigene approaches) may represent the most fundamental means of ablating gene function. Two mechanisms by which gene-specific inhibition of transcription could be achieved in vivo are oligomer binding in the major groove of duplex DNA to form a triple helix (3-5) or by strand-invasion of duplex DNA, resulting in the formation of a stable D-loop [using peptide nucleic acids (PNAs) (6-10)]. In the case of triplex formation, recognition is mediated by Hoogsteen base pairing (11). The nonphysiologic conditions necessary to promote third-strand binding of an oligomer to a duplex target has largely precluded the use of this approach for in vivo studies. However, the requirement for low pH or specific ionic conditions to stabilize cytosine protonation (12, 13) has recently been obviated by pH-independent cytosine surrogates (14, 15).PNAs consist of achiral monomer subunits derived from N-ethylaminoglycine bearing appendent nucleoside heterocyclic bases, which are coupled by standard methods of amide bond formation. We and others (6, 9, 10) have shown that thymidine-rich PNAs recognize complimentary sequences in duplex DNA by strand-invasion, which results inThe publication costs of this article were defrayed in part by page charge payment. This article must therefore ...

The antisense activity and gene specificity of two classes of oligonucleotides (ONs) were directly compared in a highly controlled assay. One class of ONs has been proposed to act by targeting the degradation of specific RNAs through an RNase H-mediated mechanism and consists of C-5 propynyl pyrimidine phosphorothioate ONs (propyne-S-ON). The second class of antisense agents has been proposed to function by sterically blocking target RNA formation, transport or translation and includes sugar modified (2'-O-allyl) ONs and peptide nucleic acids (PNAs). Using a CV-1 cell based microinjection assay, we targeted antisense agents representing both classes to various cloned sequences localized within the SV40 large T antigen RNA. We determined the propyne-S-ON was the most potent and gene-specific agent of the two classes which likely reflected its ability to allow RNase H cleavage of its target. The PNA oligomer inhibited T Ag expression via an antisense mechanism, but was less effective than the propyne-S-ON; the lack of potency may have been due in part to the PNAs slow kinetics of RNA association. Interestingly, unlike the 2'-O-allyl ON, the antisense activity of the PNA was not restricted to the 5' untranslated region of the T Ag RNA. Based on these findings we conclude that PNAs could be effective antisense agents with additional chemical modification that will lead to more rapid association with their RNA target.