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 ...
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.
Residue F4 (Phe 97) undergoes the most dramatic ligand-linked transition in Scapharca dimeric hemoglobin, with its packing in the heme pocket in the unliganded (T) state suggested to be a primary determinant of its low affinity. Mutation of Phe 97 to Leu (previously reported), Val, and Tyr increases oxygen affinity from 8- to 100-fold over that of the wild type. The crystal structures of F97L and F97V show side chain packing in the heme pocket for both R and T state structures. In contrast, in the highest-affinity mutation, F97Y, the tyrosine side chain remains in the interface (high-affinity conformation) even in the unliganded state. Comparison of these mutations reveals a correlation between side chain packing in the heme pocket and oxygen affinity, indicating that greater mass in the heme pocket lowers oxygen affinity due to impaired movement of the heme iron into the heme plane. The results indicate that a key hydrogen bond, previously hypothesized to have a central role in regulation of oxygen affinity, plays at most only a small role in dictating ligand affinity. Equivalent mutations in sperm whale myoglobin alter ligand affinity by only 5-fold. The dramatically different responses to mutations at the F4 position result from subtle, but functionally critical, stereochemical differences. In myoglobin, an eclipsed orientation of the proximal His relative to the A and C pyrrole nitrogen atoms provides a significant barrier for high-affinity ligand binding. In contrast, the staggered orientation of the proximal histidine found in liganded HbI renders its ligand affinity much more susceptible to packing contacts between F4 and the heme group. These results highlight very different strategies used by cooperative hemoglobins in molluscs and mammals to control ligand affinity by modulation of the stereochemistry on the proximal side of the heme.
Peptide nucleic acids (PNA) are oligodeoxynucleotide (ODN) analogs in which the sugar phosphate backbone of the ODN has been replaced by one derived from units of N-ethylaminoglycine. PNAs recognize DNA and RNA in a sequence specific manner and form complexes that can be characterized by biophysical methods. The binding motif i s context dependent; homopyrimidine PNAs combine with complementary polypurine targets to form stoichiometric 2:l complexes, whereas PNAs containing both purine and pyrimidine bases afford a 1 :I heteroduplex with rnis-match sensitivity comparable to that found in dsDNA. These complexes mediate the antigene and antisense effects of PNAs via the steric blockade of enzyme complexes responsible for DNA transcription, cDNA synthesis, and RNA translation. PNAs, like ODNs, are taken up by cells via endocytosis leading to their entrapment within intracytoplasmic vesicles. Under circumstances where agent delivery i s solved by cell microinjection, PNAs can effect selective inhibition of endogenous and exogenous genes. The impact of biophysical parameters on the biological assessment of PNAs as antisense inhibitors of gene expression i s presented and discussed. Q 1995 Wiley-Liss, Inc.
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