DNA Splicing Research Articles

Developmentally programmed DNA splicing in Paramecium reveals short-distance crosstalk between DNA cleavage sites

Somatic genome assembly in the ciliate Paramecium involves the precise excision of thousands of short internal eliminated sequences (IESs) that are scattered throughout the germline genome and often interrupt open reading frames. Excision is initiated by double-strand breaks centered on the TA dinucleotides that are conserved at each IES boundary, but the factors that drive cleavage site recognition remain unknown. A degenerate consensus was identified previously at IES ends and genetic analyses confirmed the participation of their nucleotide sequence in efficient excision. Even for wild-type IESs, however, variant excision patterns (excised or nonexcised) may be inherited maternally through sexual events, in a homology-dependent manner. We show here that this maternal epigenetic control interferes with the targeting of DNA breaks at IES ends. Furthermore, we demonstrate that a mutation in the TA at one end of an IES impairs DNA cleavage not only at the mutant end but also at the wild-type end. We conclude that crosstalk between both ends takes place prior to their cleavage and propose that the ability of an IES to adopt an excision-prone conformation depends on the combination of its nucleotide sequence and of additional determinants.

Sperm-mediated 'reverse' gene transfer: a role of reverse transcriptase in the generation of new genetic information

Sperm-mediated gene transfer (SMGT) is a procedure through which new genetic traits are introduced in animals by exploiting the ability of spermatozoa to take up exogenous DNA molecules and deliver them to oocytes at fertilization. The interaction of exogenous DNA with sperm cells is a regulated process mediated by specific factors; among those, a reverse transcriptase (RT) activity plays a central role in SMGT. 'Retro-genes' are generated either through reverse transcription of exogenous RNA internalized in spermatozoa, or through sequential transcription, splicing and reverse transcription of exogenous DNA. The resulting retro-genes are delivered to oocytes and transmitted to embryos and born animals as low-copy, transcriptionally competent, extrachromosomal structures capable of determining new phenotypic traits. Retro-genes can be further transmitted through sexual reproduction from founders to their F1 progeny: new genetic and phenotypic features, unlinked to chromosomes, can thus be generated and inherited in a non-Mendelian ratio. We have called this phenomenon sperm-mediated 'reverse' gene transfer (SMRGT). Thus, a RT-mediated machinery operates in sperm cells and is responsible for the genesis and non-Mendelian propagation of new genetic information. The features of RT-generated traits elicited in SMRGT resemble those characterized in recent studies of RNA-mediated inheritance of extra-genomic information.

Rapid Assembly of Multiple-Exon cDNA Directly from Genomic DNA

BackgroundPolymerase chain reaction (PCR) is extensively applied in gene cloning. But due to the existence of introns, low copy number of particular genes and high complexity of the eukaryotic genome, it is usually impossible to amplify and clone a gene as a full-length sequence directly from the genome by ordinary PCR based techniques. Cloning of cDNA instead of genomic DNA involves multiple steps: harvest of tissues that express the gene of interest, RNA isolation, cDNA synthesis (reverse transcription), and PCR amplification. To simplify the cloning procedures and avoid the problems caused by ubiquitously distributed durable RNases, we have developed a novel strategy allowing the cloning of any cDNA or open reading frame (ORF) with wild type sequence in any spliced form from a single genomic DNA preparation.MethodologyOur “Genomic DNA Splicing” technique contains the following steps: first, all exons of the gene are amplified from a genomic DNA preparation, using software-optimized, highly efficient primers residing in flanking introns. Next, the tissue-specific exon sequences are assembled into one full-length sequence by overlapping PCR with deliberately designed primers located at the splicing sites. Finally, software-optimized outmost primers are exploited for efficient amplification of the assembled full-length products.ConclusionsThe “Genomic DNA Splicing” protocol avoids RNA preparation and reverse transcription steps, and the entire assembly process can be finished within hours. Since genomic DNA is more stable than RNA, it may be a more practical cloning strategy for many genes, especially the ones that are very large and difficult to generate a full length cDNA using oligo-dT primed reverse transcription. With this technique, we successfully cloned the full-length wild type coding sequence of human polymeric immunoglobulin receptor, which is 2295 bp in length and composed of 10 exons.

DNA splicing: computing by observing

Motivated by several techniques for observing molecular processes in real-time we introduce a computing device that stresses the role of the observer in biological computations and that is based on the observed behavior of a splicing system. The basic idea is to introduce a marked DNA strand into a test tube with other DNA strands and restriction enzymes. Under the action of these enzymes the DNA starts to splice. An external observer monitors and registers the evolution of the marked DNA strand. The input marked DNA strand is then accepted if its observed evolution follows a certain expected pattern. We prove that using simple observers (finite automata), applied on finite splicing systems (finite set of rules and finite set of axioms), the class of recursively enumerable languages can be recognized.

Epigenetics in inflammatory rheumatic diseases

Inflammatory rheumatic disorders, such as rheumatoid arthritis (RA) and connective tissue diseases, are characterized by chronic inflammation that generally can be overcome by lifelong administration of immunosuppressive therapies. In most of these diseases, factors of genetic predisposition have been described, in particular the influence of distinct HLA haplotypes. In addition, environmental factors, including nutrition, infection, and exposure to sunlight, have been postulated as disease-driving agents. It is, however, unclear how genetic susceptibility in concert with the factors mentioned lead to the development of disease in one individual but not in another. The successful accomplishment of the Human Genome Project, which has yielded sequences for !25,000 identified human genes, presents a challenge to biomolecular research, since most of the identified genes code for biologic functions that have yet to be discovered. The functional characterization of these genes in normal physiologic processes as well as in the pathogenesis of diseases, a concept generally referred to as functional genomics (1), remains the main issue for biomedical research in the coming years. Within the nucleus of a human cell, the DNA sequence contains !3 billion basepairs, covering most of the nonheterochromatic portions of the genome. The low number of genes detected by the Human Genome Project is, however, most surprising. Moreover, between humans and chimpanzees, a remarkable genetic similarity of nearly 99% has been reported, thus highlighting the importance of distinguishing areas of the genome that would define genetic changes unique to humans (2). The low number of genes reflects the functional impact of posttranscriptional as well as posttranslational modifications and of epigenetic alterations in creating proteome diversity. Epigenetic alterations comprise heritable modifications of the DNA without any change in the base sequence of the genetic code per se. Two major groups of epigenetic alterations have been identified, i.e., DNA methylation and histone modifications. Changes to DNA or alternative splicing of messenger RNA (mRNA) transcripts has a strong impact on proteome complexity, and thus the utility of genomic information is somewhat restricted by the lack of precision in predicting genes, gene structures, and alternative splices (3,4). Epigenetic alterations can be inherited; however, changes can occur over the full span of a human life. When analyzing the epigenome (the genome-wide distribution of epigenetic alterations) in homozygous twins, Farga et al found a very similar epigenetic pattern in younger twin pairs, whereas older twin pairs showed several differences in the pattern of histone acetylation and DNA methylation, particularly in those whose lifestyles were not shared (5). Currently, the epigenome is not measured systematically, and epigenetic changes have not been assessed within the Human Genome Project. For several illnesses, however, evidence indicates that they are linked to specific epigenetic processes and variations, which might provide an explanation for the late onset and strong age dependency of some diseases or the progressive nature of many common diseases (6). Moreover, it is likely that epigenetic alterations underpin the role of environmental factors in the pathogenesis of diseases in genetically susceptible individuals, and thus might broaden the scope of design of new therapeutic strategies. In this review article, we focus on the emerging concepts of epigenetics (summarized in Figure 1) as they apply to inflammatory rheumatic diseases.

Isolation and identification of AIDA-I receptors in porcine intestinal mucus

Porcine AIDA-I positive Escherichia coli causes diarrhea in neonatal piglets and AIDA-I adhesin is an important virulence factor involved in intestinal colonization with biofilm formation. This biofilm consists of AIDA-I + E. coli bacteria stratified within mucus layers covering the intestinal mucosa. Based on the intimate interaction between AIDA-I + E. coli and mucus within the intestinal biofilm, we hypothesized that porcine intestinal mucus contains receptor(s) for AIDA-I adhesin. Since porcine AIDA-I receptors have not been identified, we employed affinity chromatography and in vitro adhesion assays to investigate AIDA-I binding proteins in porcine intestinal mucus that might serve as receptors for attachment of AIDA-I positive E. coli. We demonstrated that porcine mucus contains 65 and 120 kDa proteins (p65 and p120) that bind with high affinity to purified AIDA-I adhesin and that AIDA-I positive E. coli binds to these proteins with higher affinity than do AIDA-I negative mutant. The identity of p65 was not determined based on LC–MS/MS data, whereas p120 was matched to two nuclear proteins (namely, DNA damage binding protein and splicing factor 3b) and one cytoplasmic protein, which is an IgG Fc binding protein. Based on similar amino acid homology, molecular weight, structural similarity to mucin and reported evidence of being secreted by goblet cells into the intestinal lumen, we think that the IgG Fc binding protein is most likely candidate to serve as a potential receptor in intestinal mucus for AIDA-I adhesin.

Circulating Methylated DNA: A New Generation of Tumor Markers

In this issue of Clinical Cancer Research , Wallner et al. suggest that methylated DNA released in the circulation could possibly be used as a prognostic indicator and for tumor detection ([1][1]). Tumor markers are biological substances that indicate the presence of cancer. They are usually

Breast Tumor Susceptibility to Chemotherapy Via Support Vector Machines

Support vector machine (SVM) classification together with alternative DNA splicing techniques were used to generate a classifier for breast cancer patients that are partial-responders to chemotherapy treatment. Partial responders are patients whose tumors were reduced by at least 50%. A stand-alone linear-programming-based SVM algorithm was used to separate the partial-responders from the nonresponders. A novel aspect of the classification approach utilized here is that each patient is represented by multiple points (replicates) in the 25-dimensional input space of DNA splice measurements.Replicates for all patients except those for one patient, were used as a training set. The average of the replicates for the patient left out was then used to test the leave one out correctness (looc). The looc for a group of 35 patients, with 9 partial-responders and 26 nonresponders was 88.6%, in an input space of 5 gene expressions extracted from an original space of 25 gene expression transcripts.

Drosophila SPF45: A Bifunctional Protein with Roles in Both Splicing and DNA Repair

The sequence of the SPF45 protein is significantly conserved, yet functional studies have identified it as a splicing factor in animal cells and as a DNA-repair protein in plants. Using a combined genetic and biochemical approach to investigate this apparent functional discrepancy, we unify and validate both of these studies by demonstrating that the Drosophila melanogaster protein is bifunctional, with independent functions in DNA repair and splicing. We find that SPF45 associates with the U2 snRNP and that mutations that remove the C-terminal end of the protein disrupt this interaction. Although animals carrying this mutation are viable, they are nevertheless compromised in their ability to regulate Sex-lethal splicing, demonstrating that Sex-lethal is an important physiological target of SPF45. Furthermore, these mutant animals exhibit phenotypes diagnostic of difficulties in recovering from exogenously induced DNA damage. The conclusion that SPF45 functions in the DNA-repair pathway is strengthened by finding both genetic and physical interactions between SPF45 and RAD201, a previously uncharacterized member of the RecA/Rad51 protein family. Together with our finding that the fly SPF45 protein increases the survival rate of mutagen-treated bacteria lacking the RecG helicase, these studies provide the tantalizing suggestion that SPF45 has an ancient and evolutionarily conserved role in DNA repair.

Description of a PCR-based technique for DNA splicing and mutagenesis by producing 5' overhangs with run through stop DNA synthesis utilizing Ara-C

BackgroundSplicing of DNA molecules is an important task in molecular biology that facilitates cloning, mutagenesis and creation of chimeric genes. Mutagenesis and DNA splicing techniques exist, some requiring restriction enzymes, and others utilize staggered reannealing approaches.ResultsA method for DNA splicing and mutagenesis without restriction enzymes is described. The method is based on mild template-dependent polymerization arrest with two molecules of cytosine arabinose (Ara-C) incorporated into PCR primers. Two rounds of PCR are employed: the first PCR produces 5' overhangs that are utilized for DNA splicing. The second PCR is based on polymerization running through the Ara-C molecules to produce the desired final product. To illustrate application of the run through stop mutagenesis and DNA splicing technique, we have carried out splicing of two segments of the human cofilin 1 gene and introduced a mutational deletion into the product.ConclusionWe have demonstrated the utility of a new PCR-based method for carrying out DNA splicing and mutagenesis by incorporating Ara-C into the PCR primers.

Interaction of U-box E3 ligase SNEV with PSMB4, the β7 subunit of the 20 S proteasome

Recognition of specific substrates for degradation by the ubiquitin-proteasome pathway is ensured by a cascade of ubiquitin transferases E1, E2 and E3. The mechanism by which the target proteins are transported to the proteasome is not clear, but two yeast E3s and one mammalian E3 ligase seem to be involved in the delivery of targets to the proteasome, by escorting them and by binding to the 19 S regulatory particle of the proteasome. In the present study, we show that SNEV (senescence evasion factor), a protein with in vitro E3 ligase activity, which is also involved in DNA repair and splicing, associates with the proteasome by directly binding to the beta7 subunit of the 20 S proteasome. Upon inhibition of proteasome activity, SNEV does not accumulate within the cells although its co-localization with the proteasome increases significantly. Since immunofluorescence microscopy also shows increased co-localization of SNEV with ubiquitin after proteasome inhibition, without SNEV being ubiquitinated by itself, we suggest that SNEV shows E3 ligase activity not only in vitro but also in vivo and escorts its substrate to the proteasome. Since the yeast homologue of SNEV, Prp19, also interacts with the yeast beta7 subunit of the proteasome, this mechanism seems to be conserved during evolution. Therefore these results support the hypothesis that E3 ligases might generally be involved in substrate transport to the proteasome. Additionally, our results provide the first evidence for a physical link between components of the ubiquitin-proteasome system and the spliceosome.

Identification of Interaction Partners and Substrates of the Cyclin A1-CDK2 Complex

The CDK2-associated cyclin A1 is essential for spermatogenesis and contributes to leukemogenesis. The detailed molecular functions of cyclin A1 remain unclear, since the molecular networks involving cyclin A1-CDK2 have not been elucidated. Here, we identified novel cyclin A1/CDK2 interaction partners in a yeast triple-hybrid approach. Several novel proteins (INCA1, KARCA1, and PROCA1) as well as the known proteins GPS2 (G-protein pathway suppressor 2), Ku70, receptor for activated protein kinase C1/guanine nucleotide-binding protein beta-2-like-1, and mRNA-binding motif protein 4 were identified as interaction partners. These proteins link the cyclin A1-CDK2 complex to diverse cellular processes such as DNA repair, signaling, and splicing. Interactions were confirmed by GST pull-down assays and co-immunoprecipitation. We cloned and characterized the most frequently isolated unknown gene, which we named INCA1 (inhibitor of CDK interacting with cyclin A1). The nuclear INCA1 protein is evolutionarily conserved and lacks homology to any known gene. This novel protein and two other interacting partners served as substrates for the cyclin A1-CDK2 kinase complex. Cyclin A1 and all interaction partners were highly expressed in testis with varying degrees of tissue specificity. The highest expression levels were observed at different time points during testis maturation, whereas expression levels in germ cell cancers and infertile testes decreased. Taken together, we identified testicular interaction partners of the cyclin A1-CDK2 complex and studied their expression pattern in normal organs, testis development, and testicular malignancies. Thereby, we establish a new basis for future functional analyses of cyclin A1. We provide evidence that the cyclin A1-CDK2 complex plays a role in several signaling pathways important for cell cycle control and meiosis.

Genome remodeling in ciliated protozoa.

The germline genomes of ciliated protozoa are dynamic structures, undergoing massive DNA rearrangement during the formation of a functional macronucleus. Macronuclear development involves chromosome fragmentation coupled with de novo telomere synthesis, numerous DNA splicing events that remove internal segments of DNA, and, in some ciliates, the reordering of scrambled gene segments. Despite the fact that all ciliates share similar forms of DNA rearrangement, there appears to be great diversity in both the nature of the rearranged DNA and the molecular mechanisms involved. Epigenetic effects on rearrangement have also been observed, and recent work suggests that chromatin differentiation plays a role in specifying DNA segments either for rearrangement or for elimination.

Virology: Overview

Virology: Overview

Five novel alternatively spliced transcripts of DNA (cytosine-5) methyltransferase 2 in human peripheral blood leukocytes

Alternative splicing of RNA molecules transcribed from DNA (cytosine-5) methyltransferases has been proposed as a mechanism by which methylation is able to effect diverse biological processes in higher eukaryotes. This study has investigated transcriptional versatility of DNA (cytosine-5) methyltransferase 2, which may methylate cytosine residues within 5′-CCTGG-3′ pentanucleotides in regions of the human genome devoid of 5′-CG-3′ methylation. Five novel splice variants of DNA (cytosine-5) methyltransferase 2 were identified in the peripheral blood leukocytes of healthy subjects following cloning and sequencing of RT-PCR products amplified using gene specific oligodeoxyribonucleotide primers. The generation of some of these splice variants may be influenced by the formation of secondary structures within pre-mRNA due to the repetition of sequences flanking alternatively spliced exons in a reverse and complementary orientation on the same strand. These findings enable novel approaches to investigate the role of RNA secondary structures in alternative splicing. The DNA (cytosine-5) methyltransferase 2 splice variants are generated in all the major cell types of peripheral blood, as well as in neoplastic lymphoid cells indicating that they are unlikely to generate proteins involved in control of the cell cycle or cellular differentiation. Interestingly, the gene products generated by some splice variants completely or partially lack highly conserved amino acid motifs shown to be important for the catalysis of cytosine methylation. The possibility cannot be excluded, therefore, that alternative splicing of DNA (cytosine-5) methyltransferase 2 pre-mRNA may generate protein isoforms which have different methylating capabilities or which are involved in biological processes other than the catalysis of cytosine methylation.

A tale of two HSV-1 helicases: Roles of phage and animal virus helicases in DNA replication and recombination

Helicases play essential roles in many important biological processes such as DNA replication, repair, recombination, transcription, splicing, and translation. Many bacteriophages and plant and animal viruses encode one or more helicases, and these enzymes have been shown to play many roles in their respective viral life cycles. In this review we concentrate primarily on the roles of helicases in DNA replication and recombination with special emphasis on the bacteriophages T4, T7, and A as model systems. We explore comparisons between these model systems and the herpesviruses--primarily herpes simplex virus. Bacteriophage utilize various pathways of recombination-dependent DNA replication during the replication of their genomes. In fact the study of recombination in the phage systems has greatly enhanced our understanding of the importance of recombination in the replication strategies of bacteria, yeast, and higher eukaryotes. The ability to "restart" the replication process after a replication fork has stalled or has become disrupted for other reasons is a critical feature in the replication of all organisms studied. Phage helicases and other recombination proteins play critical roles in the "restart" process. Parallels between DNA replication and recombination in phage and in the herpesviruses is explored. We and others have proposed that recombination plays an important role in the life cycle of the herpesviruses, and in this review, we discuss models for herpes simplex virus type 1 (HSV-1) DNA replication. HSV-1 encodes two helicases. UL9 binds specifically to the origins of replication and is believed to initiate HSV DNA replication by unwinding at the origin; the heterotrimeric helicase-primase complex, encoded by UL5, UL8, and UL52 genes, is believed to unwind duplex viral DNA at replication forks. Structure-function analyses of UL9 and the helicase-primase are discussed with attention to the roles these proteins might play during HSV replication.

Programmed DNA under-amplification in Paramecium primaurelia.

Ciliates are unicellular eukaryotic organisms that contain two types of nuclei throughout their vegetative life, transcriptionally active macronuclei governing the cell phenotype, and transcriptionally inert micronuclei. Following sexual reproduction, new macronuclear genomes regularly develop from micronuclear genomes through programmed DNA rearrangements that include DNA splicing, DNA fragmentation and DNA amplification. In the course of characterization of the micronuclear version of the 9.0 kb G gene, which encodes the G surface antigen in Paramecium primaurelia, we characterized a G gene duplicate. Compared with the G gene, the G gene duplicate displays features identifying it as a psi G pseudogene. About 1.6 kb upstream from the G gene, we characterized a new gene, the P gene. A related psi P putative pseudogene lies 1.6 kb upstream from the psi G pseudogene, showing that the duplicated region extends over > 15 kb and putatively defining it as a pseudogene region. Within macronuclear genomes, this region is highly under-amplified; its level never exceeds 20% of that of the corresponding G gene/P gene region in the 11 cell clones we tested. Under-amplification of the psi G pseudogene/psi P putative pseudogene region could be due to its distal position on macronuclear chromosomes, the use of alternative DNA fragmentation domains being frequent in Paramecium species, or to intrinsically lower amplification of a large genomic region. Therefore, the psi G pseudogene/psi P putative pseudogene region presented in this study provides a useful tool for the analysis of DNA fragmentation and/or amplification in eukaryotic genomes.

ConZA8 encodes an abundant protein targeted to the developing macronucleus in Euplotes crassus.

During macronuclear development in the ciliate Euplotes crassus, micronuclear-derived chromosomes undergo a series of rearrangements that include polytenization, DNA splicing, chromosome fragmentation, and telomere addition and processing. Although cis-acting signals that may function in the regulation of these events have been characterized, the proteins that mediate these events have not yet been identified. To identify development-specific factors that may be involved in DNA rearrangement, we previously isolated clones of a number of genes that are expressed only during early macronuclear development. Here, we report the genomic and cDNA sequences of one of these genes, conZA8. The analysis indicates that the conZA8 gene encodes a novel, 468-amino acid, proline-rich protein. Antibodies were raised against both a recombinant form of the conZA8 protein and an internal peptide. Immunoblotting and immunofluorescence analyses indicated that the conZA8 protein is highly abundant, expressed only during the polytene chromosome stage of macronuclear development, and localized to the developing macronucleus. Possible functions of the conZA8 protein are discussed.

DNA Splicing by Directed Ligation (SDL)

Splicing by directed ligation (SDL) is a method of in-phase joining of PCR-generated DNA fragments that is based on a pre-designed combination of class IIS restriction endonuclease recognition and cleavage sites. Since these enzymes cleave outside of their recognition sites, the resulting sticky end can have any desired sequence, and the site itself can be removed and does not appear in the final spliced DNA product. SDL is based on the addition of class IIS recognition sites onto primers used to amplify DNA sequences. Cleavage of the PCR products results in elimination of the recognition site-containing flanking sequences and leaves the DNA fragments crowned with protruding ends. With careful design of the sticky ends, several segments can be ligated together in a predetermined order in a single reaction. SDL requires fewer rounds of amplification than overlap extension methods, and is particularly useful for creating a series of recombinants that differ in one segment.

Improving the efficiency of a user-driven learning system with reconfigurable hardware. Application to DNA splicing.

This paper describes a new approach to problem solving by splitting up problem component parts between software and hardware. Our main idea arises from the combination of two previously published works. The first one proposed a conceptual environment of concept modelling in which the machine and the human expert interact. The second one reported an algorithm based on reconfigurable hardware system which outperforms any kind of previously published genetic data base scanning hardware or algorithms. Here we show how efficient the interaction between the machine and the expert is when the concept modelling is based on reconfigurable hardware system. Their cooperation is thus achieved with an real time interaction speed. The designed system has been partially applied to the recognition of primate splice junctions sites in genetic sequences.