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This work introduces minimum accumulative degeneracy, a variant of the degenerate primer design problem, which is particularly useful when a large number of sequences are to be covered by a set of restricted number of primers. A primer set, which is designed on a minimum accumulative degeneracy basis, especially helps to reduce nonspecific PCR amplification of undesired DNA fragments, as fewer primer species are present in PCR. A Boltzmann machine is designed to solve the minimum accumulative degeneracy degenerate primer design problem, called the MAD-DPD Boltzmann machine. This algorithm shows great flexibility, as it can be determined either to solve the problem with strict fidelity to covering all input sequences or to exclude some input sequences if it results in less degenerate primers. This Boltzmann machine is successfully implemented in designing a new set of primers for amplification of antibody variable fragments from mouse spleen cells, which theoretically covers more diverse antibody sequences than currently available primers. The MAD-DPD Boltzmann machine is available online at bioinf.cs.ipm.ir/download/MAD_DPD08172007.zip.

The yeast Mre11 protein participates in important cellular functions such as DNA repair and telomere maintenance. Analysis of structure-function relationships of Mre11 has led to identification of several separation-of-function mutations as well as N- and C-terminal domains essential for Mre11 meiotic and mitotic activities. Previous studies have established that there is a strong correlation between Mre11 DNA repair and telomere maintenance functions and that Mre11-Rad50-Xrs2 complex formation appears to be essential for both of these activities. Here we report that the mre11(ts) allele, previously shown to cause temperature-dependent defects in DNA repair and meiosis, confers a temperature-independent telomere shortening, indicating that mre11(ts) is a separation-of-function mutation with respect to DNA repair and telomere maintenance. In a yeast two-hybrid system, Mre11(ts) fails to form a homodimer or interact with Rad50 and Xrs2 irrespective of experimental temperatures. These observations collectively suggest that the Pro162Ser substitution in Mre11(ts) confers a novel separation of Mre11 mitotic functions. Moreover, we observed that while overexpression of the 5′-3′ exonuclease gene EXO1 partially complements the MMS sensitivity of mre11, rad50, and xrs2 null mutants, it has no effect on telomere shortening in these strains. This result provides additional evidence on possible involvement of distinctive mechanisms in DNA repair and telomere maintenance by the Mre11-Rad50-Xrs2 complex.

The yeast Mre11 is a multi-functional protein and is known to form a protein complex with Rad50 and Xrs2. In order to elucidate the relationship between Mre11 complex formation and its mitotic functions, and to determine domain(s) required for Mre11 protein interactions, we performed yeast two-hybrid and functional analyses with respect to Mre11 DNA repair and telomere maintenance. Evidence presented in this study indicates that the N-terminal region of Mre11 constitutes the core homo-dimerization and hetero-dimerization domain and is sufficient for Mre11 DNA repair and maintaining the wild-type telomere length. In contrast, a stretch of 134 amino acids from the extreme C-terminus, although essential for achieving a full level of self-association, is not required for the aforementioned Mre11 mitotic functions. Interestingly, deletion of these same 134 amino acids enhanced the interaction of Mre11 with Rad50 and Xrs2, which is consistent with the notion that this region is specific for meiotic functions. While Mre11 self-association alone is insufficient to provide the above mitotic activities, our results are consistent with a strong correlation between Mre11-Rad50-Xrs2 complex formation, mitotic DNA repair and telomere maintenance. This correlation was further strengthened by analyzing two mre11 phosphoesterase motif mutants ( mre11-2 and rad58S ), which are defective in DNA repair, telomere maintenance and protein interactions, and a rad50S mutant, which is normal in both complex formation and mitotic functions. Together, these results support and extend a current model regarding Mre11 structure and functions in mitosis and meiosis.

The Saccharomyces cerevisiae MRE11 gene plays an important role in meiotic recombination, mitotic DNA repair and telomere maintenance. We present the isolation of hMRE11B cDNA from a human HeLa cell cDNA library as an MRE11 homolog. Compared to the previously identified hMRE11 , hMRE11B contains an additional 84 bp sequence that results in a 28 amino-acid insertion close to the C-terminus. The expression pattern of hMRE11B in different tissues shows the presence of two mRNA species of approx. 2.6 and 7.5 kb. Overexpression of hMRE11B does not complement the alkylation sensitivity of the mre11 null and temperature-sensitive mutant strains. In this study, we examine factors that may explain this lack of complementation. First, both Northern and Western analyses rule out the lack of hMRE11B transcription and/or translation in yeast. Second, we demonstrate that hMre11B, like the yeast Mre11 protein, dimerizes in vivo in a yeast two-hybrid system. This dimerization requires the C-terminal one-third of hMre11B protein, which includes the 28 amino acids absent in hMre11. However, hMre11B does not interact with Mre11, Rad50 and Xrs2. Hence, the lack of protein–protein interaction between hMre11B and the yeast Mre11, Rad50, and Xrs2 may explain the inability of hMRE11B to complement the yeast mre11 mutants. We rule out the hypothesis that the lack of interaction and, in turn of complementation, is due to the absence of sequence homology at the C-terminal domain of hMre11B compared to the yeast Mre11. Instead, we propose that the C-terminus of hMre11B participates in protein–protein interaction and functions in a species-specific manner.

Background The aneurysm clip impact-compression model of spinal cord injury (SCI) is a standard injury model in animals that closely mimics the primary mechanism of most human injuries: acute impact and persisting compression. Its histo-pathological and behavioural outcomes are extensively similar to human SCI. To understand the distinct molecular events underlying this injury model we analyzed global mRNA abundance changes during the acute, subacute and chronic stages of a moderate to severe injury to the rat spinal cord. Results Time-series expression analyses resulted in clustering of the majority of deregulated transcripts into eight statistically significant expression profiles. Systematic application of Gene Ontology (GO) enrichment pathway analysis allowed inference of biological processes participating in SCI pathology. Temporal analysis identified events specific to and common between acute, subacute and chronic time-points. Processes common to all phases of injury include blood coagulation, cellular extravasation, leukocyte cell-cell adhesion, the integrin-mediated signaling pathway, cytokine production and secretion, neutrophil chemotaxis, phagocytosis, response to hypoxia and reactive oxygen species, angiogenesis, apoptosis, inflammatory processes and ossification. Importantly, various elements of adaptive and induced innate immune responses span, not only the acute and subacute phases, but also persist throughout the chronic phase of SCI. Induced innate responses, such as Toll-like receptor signaling, are more active during the acute phase but persist throughout the chronic phase. However, adaptive immune response processes such as B and T cell activation, proliferation, and migration, T cell differentiation, B and T cell receptor-mediated signaling, and B cell- and immunoglobulin-mediated immune response become more significant during the chronic phase. Conclusions This analysis showed that, surprisingly, the diverse series of molecular events that occur in the acute and subacute stages persist into the chronic stage of SCI. The strong agreement between our results and previous findings suggest that our analytical approach will be useful in revealing other biological processes and genes contributing to SCI pathology.

Serpins are a unique class of serine protease inhibitors that are becoming increasingly recognized as important regulators of insect defense mechanisms and developmental processes. Previously, we identified three Mamestra configurata serpins that were similar in structure to those encoded by the Manduca sexta Serpin-1 gene. To gain insight into the evolution and function of serpins in lepidopterans, we developed a bacterial artificial chromosome library and sequenced the entire M. configurata gene. The Serpin-1 gene was 28 kbp and had the capacity to encode nine serpin isoforms via alternate splicing of exons encoding variant reactive center loops onto a common scaffold. The relative abundance of each isoform was estimated by expressed sequence tag analysis and their expression patterns examined in various developmental stages and larval tissues. The organization of the M. configurata Serpin-1 gene was very similar to that of M. sexta Serpin-1; however, only the Ms Serpin-1Z (1 of 12) and the Mc Serpin-1a isoforms exhibited a high degree of similarity. Orthologs similar to this variant were also found in other lepidopterans, namely Bombyx mori and Plutella xylostella, suggesting that they are involved in a conserved biochemical process and likely represent the ancestral serpin variant. Expansion of the exon family encoding the Serpin-1 reactive centre loop region appears to be a product of recent duplication events that has given rise to different serpin repertoires in related insect taxa.

Background Different algorithms have been proposed to solve various versions of degenerate primer design problem. For one of the most general cases, multiple degenerate primer design problem, very few algorithms exist, none of them satisfying the criterion of designing low number of primers that cover high number of sequences. Besides, the present algorithms require high computation capacity and running time. Results PAMPS, the method presented in this work, usually results in a 30% reduction in the number of degenerate primers required to cover all sequences, compared to the previous algorithms. In addition, PAMPS runs up to 3500 times faster. Conclusion Due to small running time, using PAMPS allows designing degenerate primers for huge numbers of sequences. In addition, it results in fewer primers which reduces the synthesis costs and improves the amplification sensitivity.

The Saccharomyces cerevisiae ngs1-1 mutant was previously identified by its enhanced sensitivity to simple DNA-alkylating agents such as methyl methanesulfonate but not to UV. Molecular cloning and sequencing of NGS1 as a putative DNA-alkylation repair gene revealed that it isidentical to MRE11, a gene that is involved in DNA recombinational repair. In order to investigate functional domains of the Mre11 protein, nucleotide-sequence alterations of a number of mre11 mutant alleles, including ngs1-1, mre11-1 (ts), mre11-2, mre11-3 and mre11-58, were determined. Most of these mutations map to the N-terminus ofMre11, emphasizing the importance of this highly conserved domain. The ngs1-1 and mre11-3 mutants carry nonsense mutations resulting in truncated proteins. Missense mutations were found in mre11-1 (ts), mre11-2 and mre11-58, of which mre11-2 and mre11-58 mapped to the conserved phosphoesterase domains, indicating the involvement of these motifs in the formation and/or processing of DNA double-strand breaks. Finally, mitotic-recombination assays show that the mre11 delta mutation enhances inter-chromosomal recombination but decreases the intra-chromosomal deletion frequency. In addition, MRE11 appears to play different roles during spontaneous and alkylation-induced homologous mitotic recombination.