Biomedical subjects

A Beletskii

Publications and source records attributed to A Beletskii.

6 recordsLinked to original sources

PubMed2001

PNA interference mapping demonstrates functional domains in the noncoding RNA Xist.

The noncoding RNA Xist has been shown to be essential for X-chromosome inactivation and to coat the inactive X-chromosome (Xi). Thus, an important question in understanding the formation of Xi is whether the binding reaction of Xist is necessary for X-chromosome inactivation. In this article, we demonstrate the failure of X-chromosome silencing if the association of Xist with the X-chromosome is inhibited. The chromatin-binding region was functionally mapped and evaluated by using an approach for studying noncoding RNA function in living cells that we call peptide nucleic acid (PNA) interference mapping. In the reported experiments, a single 19-bp antisense cell-permeating PNA targeted against a particular region of Xist RNA caused the disruption of the Xi. The association of the Xi with macro-histone H2A is also disturbed by PNA interference mapping.

Animals↗

PubMed2001

Development of two bacterial artificial chromosome shuttle vectors for a recombination-based cloning and regulated expression of large genes in mammalian cells.

Most conditional expression vectors designed for mammalian cells have been valuable systems for studying genes of interest by regulating their expressions. The available vectors, however, are reliable for the short-length cDNA clones and not optimal for relatively long fragments of genomic DNA or long cDNAs. Here, we report the construction of two bacterial artificial chromosome (BAC) vectors, capable of harboring large inserts and shuttling among Escherichia coli, yeast, and mammalian cells. These two vectors, pEYMT and pEYMI, contain conditional expression systems which are designed to be regulated by tetracycline and mouse interferons, respectively. To test the properties of the vectors, we cloned in both vectors the green fluorescence protein (GFP) through an in vitro ligation reaction and the 17.8-kb-long X-inactive-specific transcript (Xist) cDNA through homologous recombination in yeast. Subsequently, we characterized their regulated expression properties using real-time quantitative RT-PCR (TaqMan) and RNA-fluorescent in situ hybridization (FISH). We demonstrate that these two BAC vectors are good systems for recombination-based cloning and regulated expression of large genes in mammalian cells.

3T3 Cells↗

PubMed2001

Transcription-induced cytosine-to-thymine mutations are not dependent on sequence context of the target cytosine.

We showed previously that transcription of a plasmid-borne kan allele increases C-to-T mutations in the nontranscribed strand. Using two new plasmid-borne kan alleles, one cmp allele, and a chromosomal kan allele, we found in this study that transcription-induced mutations are not limited to specific genes, alleles, or locations and are likely to be a general property of transcript elongation in Escherichia coli.

Alleles↗

PubMed2000

Mutations induced by bacteriophage T7 RNA polymerase and their effects on the composition of the T7 genome.

We show here that transcription by the bacteriophage T7 RNA polymerase increases the deamination of cytosine bases in the non-transcribed strand to uracil, causing C to T mutations in that strand. Under optimal conditions, the mutation frequency increases about fivefold over background, and is similar to that seen with the Escherichia coli RNA polymerase. Further, we found that a mutant T7 RNA polymerase with a slower rate of elongation caused more cytosine deaminations than its wild-type parent. These results suggest that promoting cytosine deamination in the non-transcribed strand is a general property of transcription in E. coli and is dependent on the length of time the transcription bubble stays open during elongation. To see if transcription-induced mutations have influenced the evolution of bacteriophage T7, we analyzed its genome for a bias in base composition. Our analysis showed a significant excess of thymine over cytosine bases in the highly transcribed regions of the genome. Moreover, the average value of this bias correlated well with the levels of transcription of different genomic regions. Our results indicate that transcription-induced mutations have altered the composition of bacteriophage T7 genome and suggest that this may be a significant force in genome evolution.

Amination↗

PubMed1996

Transcription-induced mutations: increase in C to T mutations in the nontranscribed strand during transcription in Escherichia coli.

Cytosines in single-stranded DNA deaminate to uracils at 140 times the rate for cytosines in double-stranded DNA. If resulting uracils are not replaced with cytosine, C to T mutations occur. These facts suggest that cellular processes such as transcription that create single-stranded DNA should promote C to T mutations. We tested this hypothesis with the Escherichia coli tac promoter and found that induction of transcription causes approximately 4-fold increase in the frequency of C to U or 5-methylcytosine to T deaminations in the nontranscribed strand. Excess mutations caused by C to U deaminations were reduced, but not eliminated, by uracil-DNA glycosylase. Similarly, mutations caused by 5-methylcytosine to T deaminations were only partially reduced by the very short-patch repair process in E.coli. These effects are unlikely to be caused by differential repair of the two strands, and our results suggest that all actively transcribed genes in E. coli should acquire more C to T mutations in the nontranscribed strand.

Base Sequence↗

PubMedDate not supplied

Correlation between transcription and C to T mutations in the non-transcribed DNA strand.

We recently showed that transcription can promote C to T mutations in the non-transcribed strand in E. coli. To study the relationship between the level of transcription and mutant frequency, an inactive allele of the kanamycin-resistance gene was expressed under the control of a hybrid promoter consisting of an UP element and the tac promoter. When this promoter is induced, the frequency of C to T mutations in the non-transcribed strand increases in rough proportion to the amount of mRNA. At the highest level of transcription at which cell growth is not affected, there is about a 10-fold increase in the frequency of mutations. This result is consistent with the hypothesis that transcription forces the non-transcribed strand to be in a single-stranded state and that this results in frequent C to T mutations.

Cell Line↗