A method for the preparation of normalized cDNA libraries enriched with full-length sequences

We developed a new method for the preparation of normalized cDNA libraries enriched with full-length sequences. It is based on the properties of the recently characterized duplex-specific nuclease from the hepatopancreas of the Kamchatka crab. The duplex-specific nuclease is thermostable, it effectively cleaves double-stranded DNA and is inactive toward single-stranded DNA (Shagin et al., Genome Res., 2002, vol. 12, pp. 1935-1942). Our method enables the normalization of cDNA samples enriched with full-length sequences without use of laborious and ineffective stages of physical separation. The efficiency of the method was demonstrated in model experiments using cDNA samples from several human tissues.     

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. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Primary Structures of Actinoporins from Sea Anemone <Emphasis Type="Italic">Oulactis orientalis</Emphasis>

Partial amino acid sequences of the actinoporins Or-A (136 aa) and Or-G (144 aa) isolated from the Sea of Japan sea anemone Oulactis orientalis were determined by sequencing the clones obtained by the amplification of genomic DNA and cDNA with primers specific to the N-terminal regions of the O. orientalis actinoporin sequences and to the C-terminal region, which is highly conservative in all the known actinoporin sequences. The complete structures of Or-A (165 aa) and Or-G (173 aa) were elucidated by sequencing the cDNA clones obtained by the rapid amplification of 3′-ends of cDNA. A comparative analysis of the amino acid sequences of the Oulactis actinoporins with those of actinoporins from tropical species revealed considerable differences in the structures of their N-terminal fragments and their membrane-binding sites. We believe that these differences could explain the lower hemolytic activities of Or-A and Or-G compared to those of actinoporins from the tropical species.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 357–362.Original Russian Text Copyright © 2005 by Il’ina, Monastyrnaya, Isaeva, Guzev, Rasskazov, Kozlovskaya     

The <Emphasis Type="Italic">NOLA2</Emphasis> and <Emphasis Type="Italic">RPS3A</Emphasis> genes as highly informative markers of human squamous cell carcinoma of lung

cDNA libraries enriched with sequences that are differentially transcribed in normal and tumor tissues were prepared using the subtractive hybridization of mixtures of cDNAs from ten patients with squamous cell carcinoma (lung cancer) and the corresponding mixtures of cDNAs from normal tissues of the same patients. An analysis of the libraries revealed two genes, NOLA2 and RPS3A, whose expression in patients with squamous cell carcinoma increased by 70%. A high frequency of enhanced expression of these genes in the cancer makes them highly informative markers of squamous cell carcinoma, which, together with other markers, can be used for reliable diagnosis of the disease.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 195–199.Original Russian Text Copyright © 2005 by Slizhikova, Vinogradova, Sverdlov.     

Spectral diversity among members of the green fluorescent protein family in hydroid jellyfish (Cnidaria,Hydrozoa)

The cDNAs encoding the genes of new proteins, homologous to the well-known Green Fluorescent Protein (GFP) from the hydroid jellyfish Aequorea victoria, were cloned. Two green fluorescent proteins from one unidentified anthomedusa, a yellow fluorescent protein from Phialidium sp., and a nonfluorescent chromoprotein from another unidentified anthomedusa were characterized. Thus, a broad diversity of GFP-like proteins among the organisms of the class Hydrozoa in both spectral properties and primary structure was shown.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 49–53.Original Russian Text Copyright © 2005 by Yanushevich, Shagin, Fradkov, Shakhbazov, Barsova, Gurskaya, Labas, Matz, K. Lukyanov, S. Lukyanov.     

Nuclease Biosynthesis and Growth of <Emphasis Type="Italic">Serratia marcescens</Emphasis> in the Presence of 2-(<Emphasis Type="Italic">p</Emphasis>-Aminobenzenesulfonamide)-thiazole

The biosynthesis of nuclease in Serratia marcescens has been studied under the conditions of purine synthesis inhibition with 2-(p-aminobenzenesulfonamide)-thiazole. The addition of this sulfonamide to S. marcescens at different growth stages is found to inhibit both culture growth and nuclease synthesis.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 365–369.Original Russian Text Copyright © 2005 by Starshinova, Filimonova.     

Construction and characterization of normalized cDNA library of maize inbred MO17 from multiple tissues and developmental stages

A comprehensive complementary DNA (cDNA) library is a valuable resource for functional genomics. In this study, we set up a normalized cDNA library of Mo17 (MONL) by saturation hybridization with genomic DNA, which contained expressed genes of eight tissues and organs from inbred Mo17 of maize (Zea mays L.). In this library, the insert sizes range from 0.4 kb to 4 kb and the average size is 1.18 kb. 10 830 clones were spotted on nylon membrane to make a cDNA microarray. Randomly picked 300 clones from the cDNA library were sequenced. The cDNA microarray was hybridized with pooled tissue mRNA probes or housekeeping gene cDNA probes. The results showed the normalized cDNA library comprehensively includes tissue-specific genes in which 71% are unique ESTs (expressed sequence tags) based on the 300 sequences analyzed. Using the BLAST program to compare the sequences against online nucleotide databases, 88% sequences were found in ZmDB or NCBI, and 12% sequences were not found in existing nucleotide databases. More than 73% sequences are of unknown function. The library could be extensively used in developing DNA markers, sequencing ESTs, mining new genes, identifying positional cloning and candidate genes, and developing microarrays in maize genomics research.__________From Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 198–206.Original English Text Copyright © 2005 by Z. Zhang, F. Zhang, Tang, Pi, Zheng.This article was submitted by the authors in English.     

DNA Methyltransferases and Structural-Functional Specificity of Eukaryotic DNA Modification

Properties of the main families of mammalian, plant, and fungal DNA methyltransferases are considered. Structural-functional specificity of eukaryotic genome sequences methylated by DNA methyltransferases is characterized. The total methylation of cytosine in DNA sequences is described, as well as its relation with RNA interference. Mechanisms of regulation of expression and modulation of DNA methyltransferase activity in the eukaryotic cell are discussed.__________Translated from Biokhimiya, Vol. 70, No. 7, 2005, pp. 885–899.Original Russian Text Copyright © 2005 by Buryanov, Shevchuk.This article was not published in the journal special issue devoted to the 70th anniversary of B. F. Vanyushin (Biochemistry (Moscow) (2005) 70, No. 5) because of limiting volume of the journal.     

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.     

Identification and Characterization <Emphasis Type="Italic">in silico</Emphasis> of Meiotic DNA

A method of in silico search for specific repetitive DNA sequences related to the synaptonemal complex (meiDNA) in mammalian genomes was developed. A study of the distribution of these repeats over chromosomes revealed their scarcity on the Y chromosome and a decrease in recombination frequency in regions enriched in meiDNA. The results are discussed in context of the model of the looplike meiotic chromosome organization during the formation of the synaptonemal complex.__________Translated from Genetika, Vol. 41, No. 5, 2005, pp. 697–701.Original Russian Text Copyright © 2005 by Grishaeva, Dadashev, Bogdanov.     

Renaturation, activation, and practical use of recombinant duplex-specific nuclease from Kamchatka crab

We overexpressed duplex-specific nuclease (DSN) from Kamchatka crab in Escherichia coli cells and developed procedures for purification, renaturation, and activation of this protein. We demonstrated identity of the properties of the native and recombinant DSN. We also successfully applied the recombinant DSN for full-length cDNA library normalization.     

High-affinity microtubule protein-higher organism DNA complexes. Many-fold enrichment in repetitive mouse DNA sequences comprised of satellite DNAs

We have examined aspects of the interaction of cycled microtubule protein preparations with ³⁵S-labeled mouse DNA tracer in a competition system with unlabelled competitor E. coli or mouse DNA. The nitrocellulose filter binding assay was used to measure interaction by scintillation counting. DNA molecular weight affected the levels of filter retained ³⁵S-labelled mouse tracer DNA. Filter retention levels increased if ³⁵S-labelled mouse DNA tracer size was increased, and the filter binding level decreased if competitor DNA size was increased. There was a sizeable, reproducible difference in the ³⁵S-labelled mouse DNA tracer binding level of about 1% when E. coli or mouse DNA competitors were compared. Mouse DNA more effectively competed with ³⁵S-labelled mouse DNA for microtubule protein binding than did E. coli DNA, suggesting that a small class of higher-organism DNA sequences interacts very strongly with microtubule protein. From other studies we know this to be the MAP fraction (Marx, K.A. and Denial, T. (1984) in The Molecular Basis of Cancer (Rein, R., ed.), Alan R. Liss, New York, in the press; and Villasante, E., Corces, V.G., Manso-Martinez, R. and Avila, J. (1981) Nucleic Acids Res. 9, 895–908). We find that this difference in competitor DNA strength is qualitatively similar under high-stringency conditions (0.5 M NaCl, high competitor [DNA]) we developed for examining high-affinity complexes. Under high-stringency conditions we isolated 1.2% and 0.6% of ³⁵S-labelled mouse DNA at 4200 and 350 bp respective sizes as nitrocellulose filter bound DNA-protein complexes. At both molecular weights these high-affinity DNA sequences, isolated from the filters, were shown to be significantly enriched in repetitive DNA sequences by S₁ nuclease solution reassociation kinetics. The kinetics are consistent with about a 4-fold mouse satellite DNA enrichment as well as enrichment in other repetitious DNA sequence classes. The high molecular weight filter-bound DNA samples were sedimented to equilibrium in CsCl buoyant density gradients and found to contain primarily mouse satellite DNA density sequences (1.691 g/cm³) with some minor fractions at other density positions (1.670, 1.682, 1.705, 1.740, 1.760 g/cm³) similar to those observed by our laboratory in previous investigations of micrococcal nuclease-resistant chromatin (Marx, K.A. (1977) Biochem. Biophys. Res. Commun. 78, 777–784). That the high-affinity microtubule-bound DNA was some 3–5-fold enriched in mouse satellite sequences was demonstrated by its characteristic BstNI restriction enzyme cleavage pattern     

A comparison of sequence complexity of nuclear and polysomal poly(A)+ RNA from different developmental stages ofXenopus laevis

Summary Nuclear poly(A)⁺ and polysomal poly(A)⁺ RNA were isolated from gastrula and early tadpole stages of the amphibianXenopus laevis. Complementary DNA was synthesized from all RNA preparations. Hybridization reactions revealed that at least all abundant and probably most of the less frequent nuclear and polysomal poly(A)⁺ RNA species present at the gastrula stage are also present at the early tadpole stage. On the other hand, there are nuclear RNA sequences at the latter stage which appear, if at all, only at lower concentrations at the gastrula stage. The polysomal poly(A)⁺ RNA hybridization reactions suggest the existence of polysomal poly(A)⁺ RNA sequences at early tadpole stages which are not present in the corresponding gastrula stage RNA.By cDNA hybridization with poly(A)^– RNA it could be shown that most of the poly(A)⁺ containing RNA sequences transcribed into cDNA were also present within the poly(A)^– RNA. It was estimated, that these sequences are 10 fold more abundant within the poly(A)^– polysomal RNA and 3–6 more abundant within the poly(A)^– nuclear RNA as compared to the poly(A)⁺ RNAs.     

Cloning and characterization of a cDNA encoding the cytosolic copper/zinc-superoxide dismutase from sweet potato tuberous root

A full-length cDNA clone encoding a putative copper/zinc-superoxide dismutase (SOD) of sweet potato, Ipomoea batatas (L.) Lam. cv Tainong 57, was isolated from a cDNA library constructed in gt10 from tuber root mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 152 amino acid residues. The deduced amino acid sequence showed higher homology (78–86%) with the sequence of the cytosolic SOD than that of the chloroplast SOD from other plant species. The residues required for coordinating copper and zinc are conserved as they are among all reported Cu/Zn-SOD sequences. In addition, it lacks recognizable plastic or mitochondrial targeting sequences. These data suggest that the isolated sweet potato clone encodes a cytosolic Cu/Zn-SOD.     

Analysis of nucleotide sequence polymorphism of internal transcribed spacers of ribosomal genes in diploid <Emphasis Type="Italic">Aegilops</Emphasis> (L.) species

The nucleotide sequence of the fragment of the internal transcribed spacer (ITS) of rDNA comprising the full-length ITS1, the gene encoding 5.8S rRNA, and part of the ITS2 sequence was determined in 22 samples of five diploid Aegilops species. The full alignment length of compared sequences was 524 bp. Species-specific substitutions were found in the ITS nucleotide sequence of rDNA of different Aegilops species. Intraspecific differences in ITS structure in diploid Aegilops species were detected for the first time. Polymorphism of the ITS nucleotide sequence within the same sample was revealed, which might be due either to differences between the genomes of individual plants comprising the sample or to the presence of several types of ribosomal genes in the genome of one plant. In general, both interspecific and intraspecific variability of the ITS nucleotide sequences of rDNA is extremely low. In total, 26 variable sites, twelve of which were informative, were identified.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 193–197.Original Russian Text Copyright © 2005 by Goryunova, Chikida, Gori, Kochieva.