Escherichia coli plasmid vectors containing synthetic translational initiation sequences and ribosome binding sites fused with the lacZ gene

The construction of a series of Escherichia coli plasmid vectors suitable for assaying the effects of gene control signals fused with the E. coli lacZ gene is reported. A synthetic deoxyoligonucleotide dodecamer 5'-CATGAATTCATG GTACTTAAGTAC-5' containing two translation initiation codons (ATG) separated by an EcoRI site was ligated with a lacZ gene derivative which lacks the codons for the first eight amino acids in plasmid pMC1403 (Casadaban et al., 1980). Two ribosome-binding sequences were synthesised and inserted into the EcoRI site before an ATG, and the effects of these sequences on lacZ gene expression in vivo measured by assaying beta-galactosidase activity. The E. coli ribosomal RNA gene (rrnB) promoter, the tetracycline resistance gene promoter, and a lambda phage promoter were cloned using these plasmids. The plasmids are 9.9 kb in size, have ampicillin resistance as a selectable marker and are generally useful for the detection and in vivo assay of gene control regions.     

Reading DNA differently

Product inhibition has provided the limiting barrier to efficient template-directed ligation and polymerization reactions. Here we review the attempts to circumvent this limitation and outline a traslation strategy that does overcome the barrier and allows the information encoded in DNA to be read and amplified into backbone-modified oligonucleotides. © 1998 John Wiley & Sons, Inc. Biopoly 48: 19–28, 1998     

Stabilization of G•C-Containing DNA Duplexes by Polyamides with Parallel Orientation in the Minor Groove

The polyamides based on 4-amino-1-methylpyrrol-2-carboxylic acid, 4-amino-1-methylimidazole-2-carboxylic acid, and -alanine that stabilize oligonucleotide duplexes consisting of GC pairs through parallel packing in the minor groove were studied. The initial duplex TTGCGCpGCGCAA melts at 28°C; the TTGCGCp[NH(CH₂)₃COPyImImNH(CH₂)₃NH(CH₃)₂][NH(CH₂)₃COImImPyNH(CH₂)₃N(CH₃)₂]GCGCAA duplex (bisphosphoramidate with parallel orientation of ligands, where Py, Im, and are the residues of 1-methyl-4-aminopyrrol-2-carboxylic and 1-methyl-4-aminoimidazole-2-carboxylic acids and -alanine, respectively), at 48°C; and the TTGCGCp[NH(CH₂)₃COImImPyNH(CH₂)₃COImImPyNH(CH₂)₃N(CH₃)₂]GCGCAA duplex (a hairpin structure with antiparallel orientation), at 56°C.     

反义核酸药物的研究现状

反义药物以其合理设计药物的可能性和精确的特异性广泛吸引了人们的注意,但反义药物的研究并非如人们最初预想的那样简单。本文从其特异性,稳定性,透过靶细胞的能力,作用强度,活性判定,给药途径,安全性和毒性,生产成本等诸方面对反义药物的研究现状,现存问题进行了综述。相信伴随这些问题的解决,反义药物很可能成为药典的一部分,给疾病的治疗带来益处。     

Heteroclinic bifurcation in a ratio-dependent predator-prey system

In this paper we study the heteroclinic bifurcation in a general ratio-dependent predator-prey system. Based on the results of heteroclinic loop obtained in [J. Math. Biol. 43(2001): 221–246], we give parametric conditions of the existence of the heteroclinic loop analytically and describe the heteroclinic bifurcation surface in the parameter space, so as to answer further the open problem raised in [J. Math. Biol. 42(2001): 489–506].Supported by NNSFC(China) # 10171071, TRAPOYT and China MOE Research Grant # 2002061003     

Reaction parameters of targeted gene repair in mammalian cells

Targeted gene repair uses short DNA oligonucleotides to direct a nucleotide exchange reaction at a designated site in a mammalian chromosome. The widespread use of this technique has been hampered by the inability of workers to achieve robust levels of correction. Here, we present a mammalian cell system in which DLD-1 cells bearing integrated copies of a mutant eGFP gene are repaired by modified single-stranded DNA oligonucleotides. We demonstrate that two independent clonal isolates, which are transcribed at different levels, are corrected at different frequencies. We confirm the evidence of a strand bias observed previously in other systems, wherein an oligonucleotide designed to be complementary to the nontranscribed strand of the target directs a higher level of repair than one targeting the transcribed strand. Higher concentrations of cell oligonucleotides in the electroporation mixture lead to higher levels of correction. When the target cell population is synchronized into S phase then released before electroporation, the correction efficiency is increased within the entire population. This model system could be useful for pharmacogenomic applications of targeted gene repair including the creation of cell lines containing single-base alterations.     

Sensitized site-specific photomodification of ssDNA by binary systems of oligonucleotide conjugates: VI. Effect of substituents in the sensitizer anthracene residue

Photomodification of ssDNA by binary systems of oligonucleotide conjugates complementary to the adjacent sequences of the target DNA was studied. One of the conjugates comprised a substituted anthracene as a sensitizer; the other,p-azidotetrafluorobenzaldehyde 3-aminopropionylhydrazone as a photoreagent. The sensitized photomodification is initiated by the 365–580-nm light through an efficient energy transfer from the photoexcitated sensitizer onto the photoreagent in a complementary complex of the binary system with the DNA target where the sensitizer and the photoreagent are sterically converged. Influence of substituents in the anthracene residue on the efficiency of the DNA sensitized photomodification was considered. The oligonucleotide conjugate of anthracene-9-al 3-aminopropionylhydrazone allows highly specific initiation of the sensitized photomodification upon irradiation with visible light at >460 nm in conditions generating no photoreaction in the sensitizer’s absence. For Part V, see [1]; prefix “d” in designations of oligonucleotides is omitted.     

Analogues of Uridinediphosphatehexoses. A New Type of Protein Glycosylation Inhibitors That Show Antiviral Activity

Abstract

A series of analogues of UDP-Glc and UDP-GlcNAc prepared by reaction of protected hexoses with ClSO₂NCO and 2′3′-O-isopropylideneuridine, inhibited glycosylation of proteins in HSV-1 infected HeLa cells and were active against several enveloped viruses.     

α-Hydroxyphosphonate Oligonucleotides: A Promising DNA Type?

Abstract

The synthesis of monomers ( S )-1, ( R )-1 and 2 derived from (5′ S )-, (5′ R )-2′-deoxythymidine-5′-C-phosphonic acids and 2′,5′-dideoxythymidine-5′-C-phosphonic acids was elaborated. The protection of the 5′-hydroxyl by the methoxycarbonyl group was a key step of the synthesis. Prepared monomers were used for the solid-phase assembly of several types oligothymidylate 15-mers ( S )-3, ( S )-4, ( S )-5, ( R )-4 and ( R )-5 containing the chiral 3′-O-P-CH(OH)-5″ internucleotide linkage. Their hybridization properties with dA₁₅ and rA₁₅ were studied as well as their resistance against nuclease cleavage.