Ekaterina A. Bogdanova

Normalization of full-length enriched cDNA

A well-recognized obstacle to efficient high-throughput analysis of cDNA libraries is the differential abundance of various transcripts in any particular cell type. Decreasing the prevalence of clones representing abundant transcripts before sequencing, using cDNA normalization, may significantly increase the efficacy of random sequencing and is essential for rare gene discovery. Duplex-specific nuclease (DSN) normalization allows the generation of normalized full-length-enriched cDNA libraries to permit a high gene discovery rate. The method is based on the unique properties of DSN from the Kamchatka crab and involves denaturation-reassociation of cDNA, degradation of the ds-fraction formed by abundant transcripts by DSN, and PCR amplification of the remaining ss-DNA fraction. The method has been evaluated in various plant and animal models.

Transcription regulation of the type II restriction-modification system AhdI

The Restriction-modification system AhdI contains two convergent transcription units, one with genes encoding methyltransferase subunits M and S and another with genes encoding the controller (C) protein and the restriction endonuclease (R). We show that AhdI transcription is controlled by two independent regulatory loops that are well-optimized to ensure successful establishment in a naïve bacterial host. Transcription from the strong MS promoter is attenuated by methylation of an AhdI site overlapping the -10 element of the promoter. Transcription from the weak CR promoter is regulated by the C protein interaction with two DNA-binding sites. The interaction with the promoter-distal high-affinity site activates transcription, while interaction with the weaker promoter-proximal site represses it. Because of high levels of cooperativity, both C protein-binding sites are always occupied in the absence of RNA polymerase, raising a question how activated transcription is achieved. We develop a mathematical model that is in quantitative agreement with the experiment and indicates that RNA polymerase outcompetes C protein from the promoter-proximal-binding site. Such an unusual mechanism leads to a very inefficient activation of the R gene transcription, which presumably helps control the level of the endonuclease in the cell.

DSN Depletion is a Simple Method to Remove Selected Transcripts from cDNA Populations

A novel DSN-depletion method allows elimination of selected sequences from full-length-enriched cDNA libraries. Depleted cDNA can be applied for subsequent EST sequencing, expression cloning, and functional screening approaches. The method employs specific features of the kamchatka crab duplex-specific nuclease (DSN). This thermostable enzyme is specific for double-stranded (ds) DNA, and is thus used for selective degradation of ds DNA in complex nucleic acids. DSN depletion is performed prior to library cloning, and includes the following steps: target cDNA is mixed with excess driver DNA (representing fragments of the genes to be eliminated), denatured, and allowed to hybridize. During hybridization, driver molecules form hybrids with the target sequences, leading to their removal from the ss DNA fraction. Next, the ds DNA fraction is hydrolyzed by DSN, and the ss fraction is amplified using long-distance PCR. DSN depletion has been tested in model experiments.

Normalization of cDNA Libraries

In a cellular transcriptome, the number of mRNA copies per gene may differ by several orders. In cDNA libraries, performed from mRNA, these proportions are the same. Normalization methods allow us to equalize numbers of gene’s copies in the library. Normalized cDNA libraries are used to discover new genes transcribed at relatively low levels or for functional screenings. Here, we observed different cDNA libraries normalization methods, which were based on hybridization (renaturation) of cDNA or DNA, or RNA. Also we described duplex-specific nuclease (DSN) normalization protocol – simple and effective cDNA libraries normalization method.

Normalizing cDNA Libraries

The characterization of rare messages in cDNA libraries is complicated by the substantial variations that exist in the abundance levels of different transcripts in cells and tissues. The equalization (normalization) of cDNA is a helpful approach for decreasing the prevalence of abundant transcripts, thereby facilitating the assessment of rare transcripts. This unit provides a method for duplex‐specific nuclease (DSN)–based normalization, which allows for the fast and reliable equalization of cDNA, thereby facilitating the generation of normalized, full‐length‐enriched cDNA libraries, and enabling efficient RNA analyses. Curr. Protoc. Mol. Biol. 90:5.12.1‐5.12.27.

Thermolabile duplex-specific nuclease.

Using random mutagenesis of the gene encoding duplex-specific nuclease from the king crab we found a new mutant that retained all properties of the wild-type protein, but exhibited a much lower thermal stability. This enzyme, denoted thermolabile duplex-specific nuclease (DSN-TL), exhibits high processivity and selective cleavage of dsDNA. The inactivation temperature for DSN-TL is 15–20°C lower than that of the widely used DNase I and shrimp nuclease, and its catalytic activity is more than 10 times higher. Moreover, DSN-TL is resistant to proteinase K treatment. These properties make DSN-TL very useful for removing genomic DNA from RNA samples intended for quantitative RT-PCR.

Is crab duplex-specific nuclease a member of the Serratia family of non-specific nucleases?

Kamchatka crab duplex-specific nuclease (Par_DSN) has been classified as a member of the family of DNA/RNA non-specific beta-beta-alpha metal finger (bba-Me-finger) nucleases, the archetype of which is the nuclease from Serratia marcescens. Although the enzyme under investigation seems to belong to the family of S. marcescens nucleases, Par_DSN exhibits a marked preference for double-stranded DNA as a substrate and this property is unusual for other members of this family. We have searched other Arthropod species and identified a number of novel Par_DSN homologs. A phylogenetic analysis demonstrates that the Par_DSN-like enzymes constitute a separate branch in the evolutionary tree of bba-Me-finger nucleases. Combining sequence analysis and site-directed mutagenesis, we found that Par_DSN and its homologs possess the nuclease domain that is slightly longer than that of classic Serratia relatives. The active site composition of Par_DSN is similar but not identical to that of classic Serratia nucleases. Based on these findings, we proposed a new classification of Par_DSN-like nucleases.

T cell immunity does not age in a long-lived rodent species

Numerous studies have demonstrated that the percentage of naïve T cells and diversity of T cell receptor (TCR) repertoire decrease with age, with some findings likewise suggesting that increased repertoire diversity may be associated with longer lifespan and healthy aging. In this work, we have analyzed peripheral TCR diversity from humans, mice, and blind mole-rats (Spalax spp.)—long-lived, hypoxia- and cancer-tolerant rodents. We employed a quantitative approach to TCR repertoire profiling based on 5’RACE with unique molecular identifiers (UMI) to achieve accurate comparison of repertoire diversity, which also required development of specific wet lab protocol and TCR gene reference for Spalax. Our direct comparison reveals a striking phenomenon. Whereas TCR diversity of mice and humans decreases with age, resulting primarily from the shrinkage of the naive T cell pool, Spalax TCR diversity remains stable even for the animals that reach extreme old age (15-17 years). This indicates that T cell immunity does not meaningfully age in long-lived rodents, at least in terms of the classical understanding of immunosenescence, which is associated with the accumulation of large numbers of memory clones. We suggest that the extraordinary longevity of Spalax may be attributable at least in part to the distinctive organization of their T cell immunity. Our findings should therefore encourage a close re-examination of the contribution of immunosenescence to life span in mammals.

SELECTIVE SUPPRESSION OF POLYMERASE CHAIN REACTION AND ITS MOST POPULAR APPLICATIONS

This chapter is devoted to methods based on suppression polymerase chain reaction (PCR) effect (cDNA library construction starting from a small amount of total RNA; suppression subtractive hybridization, SSH; ordered differential display, ODD; Marathon cDNA RACE; genome walking; variants of coincidence cloning, CC; normalization of cDNA libraries; multiplex PCR, mPCR; to in vitro cloning). Taken together, these approaches allow one to analyze complex DNA samples, from searching sequences of interest to determining complete structures of the respective genes.