Renato J. Aguilera

The C. elegans apoptotic nuclease NUC-1 is related in sequence and activity to mammalian DNase II.

The Caenorhabditis elegans nuc-1 gene has previously been implicated in programmed cell death due to the presence of persistent undegraded apoptotic DNA in nuc-1 mutant animals. In this report, we describe the cloning and characterization of nuc-1, which encodes an acidic nuclease with significant sequence similarity to mammalian DNase II. Database searches performed with human DNase II protein sequence revealed a significant similarity with the predicted C. elegans C07B5.5 ORF. Subsequent analysis of crude C. elegans protein extracts revealed that wild-type animals contained a potent endonuclease activity with a cleavage preference similar to DNase II, while nuc-1 mutant worms demonstrated a marked reduction in this nuclease activity. Sequence analysis of C07B5.5 DNA and mRNA also revealed that nuc-1(e1392), but not wild-type animals contained a nonsense mutation within the CO7B5.5 coding region. Furthermore, nuc-1 transgenic lines carrying the wild-type C07B5.5 locus demonstrated a complete complementation of the nuc-1 mutant phenotype. Our results therefore provide compelling evidence that the C07B5.5 gene encodes the NUC-1 apoptotic nuclease and that this nuclease is related in sequence and activity to DNase II.

Atomic switch networks?nanoarchitectonic design of a complex system for natural computing

Self-organized complex systems are ubiquitous in nature, and the structural complexity of these natural systems can be used as a model to design new classes of functional nanotechnology based on highly interconnected networks of interacting units. Conventional fabrication methods for electronic computing devices are subject to known scaling limits, confining the diversity of possible architectures. This work explores methods of fabricating a self-organized complex device known as an atomic switch network and discusses its potential utility in computing. Through a merger of top-down and bottom-up techniques guided by mathematical and nanoarchitectonic design principles, we have produced functional devices comprising nanoscale elements whose intrinsic nonlinear dynamics and memorization capabilities produce robust patterns of distributed activity and a capacity for nonlinear transformation of input signals when configured in the appropriate network architecture. Their operational characteristics represent a unique potential for hardware implementation of natural computation, specifically in the area of reservoir computing-a burgeoning field that investigates the computational aptitude of complex biologically inspired systems.

Characterization of an endonuclease activity which preferentially cleaves the G-rich immunoglobulin switch repeat sequences

B lymphocytes can alter selectively their immunoglobulin (Ig) isotype expressed by deletional rearrangement of the first active immunoglobulin heavy-chain (IgH) constant region (C mu) gene with one of six other constant region genes. Recombination breakpoints occur within highly repetitive switch (S) regions located upstream of each IgH constant region gene except C delta. Analysis of rearranged switch DNA junctions has not detected a consensus sequence, although the predominance of two pentamer motifs (TGGGG and TGAGC) at or near these breakpoints and throughout all murine S region sequences has led to their advocacy as the S recombination signals. In this paper, we describe the characterization and partial purification of a lymphoid-specific endo-nuclease activity which cleaves preferentially murine S region DNA. Enzyme activity selectively produced single- and double-stranded breaks at TGAGC and TGGG motifs within murine S mu and S alpha DNA. Rare cryptic cleavage sites were detected also within non-switch sequences, although cleavage intensities at these sites were reduced greatly, relative to consensus S region cleavages. Analogous activity was found in murine tissue extracts, although among the tissues assayed only spleen and thymus contained detectable activity. Subsequent biochemical characterization of this activity demonstrated that the responsible enzyme (Endo-SR) represented a previously unreported tissue-specific mammalian endonuclease. Endo-SR-specific activity could be enhanced by addition of Mg2+ or Ca2+ and inhibited by addition of Zn2+. Maximal specific activity was detected at pH 5.5 and sharply declined within +/- 0.5 pH units. In view of this enzymes sequence- and tissue-specificity, we propose that Endo-SR is a strong candidate for an endonuclease activity associated with the switch recombination process.

Combinatorial regulation of the murine RAG-2 promoter by Sp1 and distinct lymphocyte-specific transcription factors.

The recombination activation genes, RAG-1 and RAG-2, encode the critical components of the recombinase complex responsible for the generation of functional antigen receptor genes. In order to gain an insight into the transcription factors and cis-acting elements that regulate the lymphocyte-specific expression of RAG-2, the promoter-region of this gene was isolated and characterized. This analysis demonstrated that a relatively small promoter fragment could confer lymphocyte-restricted expression to a reporter construct. Our work and that of others subsequently revealed that RAG-2 promoter expression is positively regulated by BSAP (PAX-5) and c-Myb transcription factors in B- and T-lineage cells, respectively. Although BSAP and c-Myb were deemed necessary for lymphocyte-specific expression, our analysis also uncovered a G-rich region at the 5-end of the core promoter that was essential for full activity in lymphocyte cell lines. Site-directed mutagenesis revealed that a GA-box within the G-rich region was required for full promoter activity and subsequent DNA binding assays demonstrated that this element was specifically recognized by Sp1. Apart from showing that Sp1 interacts within the RAG-2 promoter, we also demonstrate that the Sp1-binding site is necessary for the high-level activation of this promoter.

Purification and characterization of the immunoglobulin switch sequence- specific endonuclease (Endo-SR) from bovine spleen

Mature B lymphocytes are able to specifically alter their Ig isotype expression in response to extracellular stimuli via a highly regulated, deletional recombination process called isotype switch recombination. Switch recombination breakpoints predominantly map to large (1-10 kb), G-rich and highly repetitive switch regions that are located directly upstream of immunoglobulin heavy-chain constant region genes. Switch region repeat structures vary considerably both within and between species, but all switch regions contain disproportionate numbers of two pentamer motifs, TGGGN and TGAGC, that are found at or directly adjacent to most analysed switch junctions. We have recently identified an endonuclease activity, Endo SR, that preferentially cleaves TGGGN and TGAGC switch motifs. We have purified the bovine endonuclease activity to homogeneity and have identified a protein with a molecular weight of approximately 32,000 that directly correlates with enzyme activity. As discussed in this report, we have found that murine and bovine Endo-SR are preferentially enriched in lymphoid tissue nuclear extracts and that both enzymes demonstrate highly similar physical and biochemical characteristics. However, each enzyme demonstrates related but distinctive specificities for consensus and degenerate TGGGN and TGAGC switch pentamer motifs.

RAG-1 AND RAG-2 GENE EXPRESSION AND V(D)J RECOMBINASE ACTIVITY ARE ENHANCED BY PROTEIN PHOSPHATASE 1 AND 2A INHIBITION IN LYMPHOCYTE CELL LINES

Expression of the recombination activating genes, RAG-1 and RAG-2, in lymphocytes, has been shown to depend on second messenger systems. An increase in intracellular cAMP upon stimulation with caffeine increases RAG expression while activation of protein kinase C (PKC) with phorbol myristate acetate (PMA) results in decreased RAG expression. The stringent regulation of recombination appears to be partially dependent on protein kinase activities which, alone, are not likely to be sufficient to regulate recombinase activity. We provide evidence implicating a role for serine/threonine phosphatases in the signal transduction pathway which regulates RAG gene expression and consequently the recombination process in lymphocytes. The cell permeable tumor promoter, calyculin-A (CLA), which is a potent inhibitor of the type 1 and 2A serine/threonine protein phosphatases (PP1 and PP2A, respectively), was shown to upregulate the expression of RAG-1 and RAG-2 in pre-B as well as mature B- and T-lymphocyte cell lines. Although agents such as caffeine known to increase intracellular cAMP levels induce RAG expression, synergy between CLA and caffeine was not detected in pre-B cells. An in vivo assessment of recombination activity after transfection of pre-B cells with an extrachromosomal recombination vector revealed a moderate increase in recombinase activity which paralleled RAG expression after CLA stimulation. Although increased cAMP levels in pre-B cells has been associated with upregulation of RAG expression we found no such upregulation in a surface immunoglobulin M positive (sIgM+) cell line, WEHI-231, and a T cell receptor positive (TCR+) murine cell line, EL-4. Moreover, in these mature lymphocyte cell lines there was no evidence of synergy in the regulation of RAG-1 and RAG-2 mRNA upon stimulation with CLA and caffeine. These results suggest novel intracellular mechanisms for the upregulation of RAG gene expression and confirm a role for type 1 and 2A phosphatases in the control of RAG gene expression and recombinase activity in lymphocyte cell lines.

Nanoarchitectonic atomic switch networks for unconventional computing

Developments in computing hardware are constrained by the operating principles of complementary metal oxide semiconductor (CMOS) technology, fabrication limits of nanometer scaled features, and difficulties in effective utilization of high density interconnects. This set of obstacles has promulgated a search for alternative, energy efficient approaches to computing inspired by natural systems including the mammalian brain. Atomic switch network (ASN) devices are a unique platform specifically developed to overcome these current barriers to realize adaptive neuromorphic technology. ASNs are composed of a massively interconnected network of atomic switches with a density of ~109 units/cm2 and are structurally reminiscent of the neocortex of the brain. ASNs possess both the intrinsic capabilities of individual memristive switches, such as memory capacity and multi-state switching, and the characteristics of large-scale complex systems, such as power-law dynamics and non-linear transformations of input signals. Here we describe the successful nanoarchitectonic fabrication of next-generation ASN devices using combined top-down and bottom-up processing and experimentally demonstrate their utility as reservoir computing hardware. Leveraging their intrinsic dynamics and transformative input/output (I/O) behavior enabled waveform regression of periodic signals in the absence of embedded algorithms, further supporting the potential utility of ASN technology as a platform for unconventional approaches to computing.

Characterization of a novel DNA binding domain within the amino‐terminal region of the RAG‐1 protein

Rag‐1 and Rag‐2 are the critical components of the V‐(D)‐J recombinase required for site‐specific recombination of the antigen receptor genes. In this study, we have examined the ability of recombinant (r) Rag‐1 and Rag‐2 to bind the recombination signal sequences (RSS) and have determined that rRag‐1, but not rRag‐2, is able to directly bind DNA. rRAG‐1 DNA binding activity was found to reside within a novel amino‐terminal arginine‐rich (RR) domain with partial homology to a variety of nucleic acid binding domains. Although the RR‐domain did not demonstrate RSS‐specificity, this DNA binding domain may stabilize the interaction of RAG‐1 with, or increase the affinity for, the V‐(D)‐J recombination signals.

Morphic atomic switch networks for beyond-Moore computing architectures

We discuss the utility of ASNs as a uniquely scalable physical platform capable of hybrid-CMOS architectures and novel computation. Through a combination of controlled design with spontaneous self-organization, an atomic switch network (ASN) has been produce as a purpose-built complex system. A highly interconnected system of Ag2S resistive switches, the ASN has been shown to produce fault-tolerant switching and a set of complex dynamics similar to biological neural networks.