Gene knockout and knockin by zinc-finger nucleases: current status and perspectives

Zinc-finger nucleases (ZFNs) are engineered site-specific DNA cleavage enzymes that may be designed to recognize long target sites and thus cut DNA with high specificity. ZFNs mediate permanent and targeted genetic alteration via induction of a double-strand break at a specific genomic site. Compared to conventional homology-based gene targeting, ZFNs can increase the targeting rate by up to 100,000-fold; gene disruption via mutagenic DNA repair is similarly efficient. The utility of ZFNs has been shown in many organisms, including insects, amphibians, plants, nematodes, and several mammals, including humans. This broad range of tractable species renders ZFNs a useful tool for improving the understanding of complex physiological systems, to produce transgenic animals, cell lines, and plants, and to treat human disease.     

A rhodopsin-like protein in Cyanophora paradoxa: gene sequence and protein immunolocalization

Here, we report the DNA sequence of the rhodopsin gene in the alga Cyanophora paradoxa (Glaucophyta). The primers were designed according to the conserved regions of prokaryotic and eukaryotic rhodopsin-like proteins deposited in the GenBank. The sequence consists of 1,272 bp comprised of 5 introns. The correspondent protein, named Cyanophopsin, showed high identity to rhodopsin-like proteins of Archea, Bacteria, Fungi, and Algae. At the N-terminal, the protein is characterized by a region with no transmembrane α-helices (80 aa), followed by a region with 7α-helices (219 aa) and a shorter 35-aa C-terminal region. The DNA sequence of the N-terminal region was expressed in E. coli and the recombinant purified peptide was used as antigen in hens to obtain polyclonal antibodies. Indirect immunofluorescence in C. paradoxa cells showed a marked labeling of the muroplast (aka cyanelle) membrane.     

Cellulose synthesis: mutational analysis and genomic perspectives using Arabidopsis thaliana

Cellulose microfibrils containing crystalline β-1,4-glucan provide the major structural framework in higher-plant cell walls. Genetic analyses of Arabidopsis thaliana now link specific genes to plant cellulose production just as was achieved some years earlier with bacteria. Cellulose-deficient mutants have defects in several members of one family within a complex glycosyltransferase superfamily and in one member of a small family of membrane-bound endo-1,4-β-glucanases. The mutants also accumulate a readily extractable β-1,4-glucan that has short chains which, in at least one case, are lipid linked. Cellulose could be made by direct extension of the glucan chain by the glycosyltransferase or, as the mutant suggests, by an indirect route which makes lipid-linked oligosaccharides. Models discussed incorporate the known enzymes and lipo-glucan and raise the possibility that different CesA glycosyltransferases may catalyse different steps. Received 5 January 2001; received after revision 25 April 2001; accepted 25 April 2001     

The V(D)J recombination activating protein RAG2 consists of a six-bladed propeller and a PHD fingerlike domain, as revealed by sequence analysis

The RAG1 and RAG2 proteins play a crucial role in V(D)J recombination by cooperating to make specific double-stranded DNA breaks at a pair of recombination signal sequences (RSSs). However, the exact function they perform has heretofore remained elusive. Using a combination of sensitive methods of sequence analysis, we show here that the active core region of the RAG2 protein, confined to the first three quarters of its sequence, is in fact composed of a six-fold repeat of a 50-residue motif which is related to the kelch/mipp motif. This motif, which forms a four-stranded twisted antiparallel β sheet, is arranged in a circular formation like blades of a propeller or turbine. Given the known properties of the β-propeller fold in mediating protein-protein interactions, it is proposed that this six-laded propeller structure of the RAG2 active core would play a crucial role in the tight complex formed by the RAG1 and RAG2 proteins and RSSs. Moreover, the presence of a plant homeodomain finger-like motif in the last quarter of the RAG2 sequence suggests a potential interaction of this domain with chromatin components. Received 6 June 1998; accepted 9 June 1998     

Structure and function of Zika virus NS5 protein: perspectives for drug design

Zika virus (ZIKV) belongs to the positive-sense single-stranded RNA-containing Flaviviridae family. Its recent outbreak and association with human diseases (e.g. neurological disorders) have raised global health concerns, and an urgency to develop a therapeutic strategy against ZIKV infection. However, there is no currently approved antiviral against ZIKV. Here we present a comprehensive overview on recent progress in structure–function investigation of ZIKV NS5 protein, the largest non-structural protein of ZIKV, which is responsible for replication of the viral genome, RNA capping and suppression of host interferon responses. Structural comparison of the N-terminal methyltransferase domain and C-terminal RNA-dependent RNA polymerase domain of ZIKV NS5 with their counterparts from related viruses provides mechanistic insights into ZIKV NS5-mediated RNA replication, and identifies residues critical for its enzymatic activities. Finally, a collection of recently identified small molecule inhibitors against ZIKV NS5 or its closely related flavivirus homologues are also discussed.     

Ca2+ release-activated Ca2+ (CRAC) current, structure, and function

Store-operated Ca²⁺ entry describes the phenomenon that connects a depletion of internal Ca²⁺ stores to an activation of plasma membrane-located Ca²⁺ selective ion channels. Tremendous progress towards the underlying molecular mechanism came with the discovery of the two respective limiting components, STIM and Orai. STIM1 represents the ER-located Ca²⁺ sensor and transmits the signal of store depletion to the plasma membrane. Here it couples to and activates Orai, the highly Ca²⁺-selective pore-forming subunit of Ca²⁺ release-activated Ca²⁺ channels. In this review, we focus on the molecular steps that these two proteins undergo from store-depletion to their coupling, the activation, and regulation of Ca²⁺ currents.     

Structural organization and cellular localization of tuftelin-interacting protein 11 (TFIP11)

Tuftelin-interacting protein (TFIP11) was first identified in a yeast two-hybrid screening as a protein interacting with tuftelin. The ubiquitous expression of TFIP11 suggested that it might have other functions in non-dental tissues. TFIP11 contains a G-patch, a protein domain believed to be involved in RNA binding. Using a green fluorescence protein tag, TFIP11 was found to locate in a novel subnuclear structure that we refer to as the TFIP body. An in vivo splicing assay demonstrated that TFIP11 is a novel splicing factor. TFIP11 diffuses from the TFIP body following RNase A treatment, suggesting that the retention of TFIP11 is RNA dependent. RNA polymerase II inhibitor (-amanitin and actinomycin D) treatment causes enlargement in size and decrease in number of TFIP bodies, suggesting that TFIP bodies perform a storage function rather than an active splicing function. The TFIP body may therefore represent a new subnuclear storage compartment for splicing components.Received 8 December 2004; received after revision 27 January 2005; accepted 8 March 2004The nucleotide sequence for the cDNA to mouse TFIP11 (previously known as TIP39 and TIP39kDa) has been submitted to Gen- Bank^TM/ EBI Data Bank with accession numbers AF290474 and NM_018783. The accession number for the human TFIP11 homologueis NM_012143.     

Chromosome analysis and meiotic behaviour ofPappogeomys (Cratogeomys)merriami merriami

Resumen Pappogeomys merriami merriami presenta 2n=36; NF=66, contando apenas con un par de acrocéntricos, siendo elGeomydae con menor número de cromosomas. Durante la meiosis existen 13 a 15 bivalentes y un complejoX-Y en tánden.     

Amino acid composition of crude and germinated guarseed flour protein (Cyamopsis Psoralioides)

Zusammenfassung Untersuchungen über die Aminosäurezusammensetzung des Mehles von ungekeimten und gekeimten Guarsamen (Cyamopsis Psoralioides), die ein billiges, hochproteinhaltiges Nahrungsmittel darstellen, haben ergeben, dass die Aminosäurekombination im gekeimten Samen einer guten Proteinqualität entspricht. Die Prüfungen über den biologischen Wert dieses Mehles sind im Gange.     

Nucleotide-binding domains of human cystic fibrosis transmembrane conductance regulator: detailed sequence analysis and three-dimensional modeling of the heterodimer

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is encoded by the gene that is defective in cystic fibrosis, the most common lethal inherited disease among the Caucasian population. CFTR belongs to the ABC transporter superfamily, whose members form macromolecular architectures composed of two membrane-spanning domains and two nucleotide-binding domains (NBDs). The experimental structures of NBDs from several ABC transporters have recently been solved, opening new avenues for understanding the structure/function relationships and the consequences of some disease-causing mutations of CFTR. Based on a detailed sequence/structure analysis, we propose here a three-dimensional model of the human CFTR NBD heterodimer. This model, which is in agreement with recent experimental data, highlights the specific features of the CFTR asymmetric active sites located at the interface between the two NBDs. Moreover, additional CFTR-specific features can be identified at the subunit interface, which may play critical roles in active site interdependence and are uncommon in other NBD dimers.Received 16 October 2003; received after revision 16 November 2003; accepted 21 November 2003