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The rat is one of the most important model organisms for biomedical and pharmacological research. However, the generation of novel models for studying specific aspects of human diseases largely depends on selection for specific traits using existing rat strains, thereby solely depending on naturally occurring variation. This study aims to provide the tools to manipulate the rat genome in a more directed way.

ICA69 is a diabetes autoantigen with no homologue of known function. Given that most diabetes autoantigens are associated with neuroendocrine secretory vesicles, we sought to determine if this is also the case for ICA69 and whether this protein participates in the process of neuroendocrine secretion. Western blot analysis of ICA69 tissue distribution in the mouse revealed a correlation between expression levels and secretory activity, with the highest expression levels in brain, pancreas, and stomach mucosa. Subcellular fractionation of mouse brain revealed that although most of the ICA69 pool is cytosolic and soluble, a subpopulation is membrane-bound and coenriched with synaptic vesicles. We used immunostaining in the HIT insulin-secreting β-cell line to show that ICA69 localizes in a punctate manner distinct from the insulin granules, suggesting an association with the synaptic-like microvesicles found in these cells. To pursue functional studies on ICA69, we chose to use the model organism Caenorhabditis elegans, for which a homologue of ICA69 exists. We show that the promoter of the C. elegansICA69 homologue is specifically expressed in all neurons and specialized secretory cells. A deletion mutant was isolated and found to exhibit resistance to the drug aldicarb (an inhibitor of acetylcholinesterase), suggesting defective neurotransmitter secretion in the mutant. On the basis of the aldicarb resistance phenotype, we named the gene ric-19(resistance to inhibitors of cholinesterase-19). The resistance to aldicarb was rescued by introducing a ric-19transgene into theric-19mutant background. This is the first study aimed at dissecting ICA69 function, and our results are consistent with the interpretation that ICA69/RIC-19 is an evolutionarily conserved cytosolic protein participating in the process of neuroendocrine secretion via association with certain secretory vesicles.

Full-length recombinant transposase Tc1A from Caenorhabditis elegans(343 amino acids) expressed in Escherichia coliBL21 in inclusion bodies has been purified in a high yield in a soluble form. The procedure includes denaturation of the inclusion bodies followed by refolding of the Tc1A protein by gel filtration. This last step is absolutely crucial to give a high yield of soluble and active protein since it allows the physical separation of the aggregates from intermediates that give rise to correctly refolded protein. This step is very sensitive to the concentration of protein. Good yields of refolded protein are obtained by refolding 2 to 12 mg of denatured protein. The other purification steps involve the initial use of gel filtration under denaturing conditions and a final step of ion-exchange chromatography. Biological activity of the purified protein was confirmed in an in vitrotransposon excision assay and its DNA-binding capacity by UV crosslinking. This new Tc1A purification procedure gives a yield of 12–16 mg/liter E. coliculture, in a form suitable for crystallization studies.

Forward genetic screens using novel assays of nematode chemotaxis to soluble compounds identified three independent transposon-insertion mutations in the gene encoding the Caenorhabditis elegansdynein heavy chain (DHC) 1b isoform. These disruptions were mapped and cloned using a newly developed PCR-based transposon display. The mutations were demonstrated to be allelic to the che-3genetic locus. This isoform of dynein shows temporally and spatially restricted expression in ciliated sensory neurons, and mutants show progressive developmental defects of the chemosensory cilia. These results are consistent with a role for this motor protein in the process of intraflagellar transport; DHC 1b acts in concert with a number of other proteins to establish and maintain the structural integrity of the ciliated sensory endings in C. elegans.

Transgenic technology is currently applied to several animal species of agricultural or medical importance, such as fish, cattle, mosquitos and parasitic worms. However, the repertoire of genetic tools used for molecular analyses of mice and Drosophilais not always applicable to other species. For example, while retroviral enhancer-trap experiments in mice can be based on embryonic stem (ES) cell technology, this is not currently an option with other animals. Similarly, the germline transformation of Drosophiladepends on the use of the P-elementtransposon, which does not jump in other genera. This article analyses the main characteristics of Tc1/marinertransposable elements, examines some of the factors that have contributed to their evolutionary success, and describes their potential, as well as their limitations, for transgenesis and insertional mutagenesis in diverse animals.

The structure of the C‐terminal DNA‐binding domain of human immunovirus‐1 integrase has been refined using nuclear magnetic resonance spectroscopy. The protein is a dimer in solution and shows a well‐defined dimer interface. The folding topology of the monomer consists of a five‐stranded β‐barrel that resembles that of Src homology 3 domains. Compared with our previously reported structure, the structure is now defined far better. The final 42 structures display a back‐bone root mean square deviation versus the average of 0.46 Å. Correlation of the structure with recent mutagenesis studies suggests two possible models for DNA binding. Proteins 1999;36:556–564. © 1999 Wiley‐Liss, Inc.

The structure of the C-terminal DNA-binding domain of human immunovirus-1 integrase has been refined using nuclear magnetic resonance spectroscopy. The protein is a dimer in solution and shows a well-defined dimer interface. The folding topology of the monomer consists of a five-stranded β-barrel that resembles that of Src homology 3 domains. Compared with our previously reported structure, the structure is now defined far better. The final 42 structures display a back-bone root mean square deviation versus the average of 0.46 Å. Correlation of the structure with recent mutagenesis studies suggests two possible models for DNA binding. Proteins 1999;36:556–564. © 1999 Wiley-Liss, Inc.

Avermectins are a class of macrocyclic lactones that is widely used in crop protection and to treat helminth infections in man and animals. Two complementary DNAs (GluClα and GluClβ) encoding chloride channels that are gated by avermectin and glutamate, respectively, were isolated from Caenorhabditis elegans. To study the role of these subunits in conferring avermectin sensitivity we isolated a mutant C. elegansstrain with a Tc1 transposable element insertion that functionally inactivated the GluClα gene (GluClα::Tc1). GluClα::Tc1 animals exhibit a normal phenotype including typical avermectin sensitivity. Xenopusoocytes expressing GluClα::Tc1 strain mRNA elicited reduced amplitude avermectin and glutamate-dependent chloride currents. Avermectin binding assays in GluClα::Tc1 strain membranes showed the presence of high affinity binding sites, with a reduced Bmax. These experiments suggest that GluClα is a target for avermectin and that additional glutamate-gated and avermectin-sensitive chloride channel subunits exist in C. elegans. We isolated a cDNA (GluClα2) encoding a chloride channel that shares 75% amino acid identity with GluClα. This subunit forms homomeric channels that are gated irreversibly by avermectin and reversibly by glutamate. GluClα2 coassembles with GluClβ to form heteromeric channels that are gated by both ligands. The presence of subunits related to GluClα may explain the low level and rarity of target site involvement in resistance to the avermectin class of compounds.

We have characterized a retrotransposon-like element (Rte-1) in C. elegans. It was identified while we were sequencing the pim related kinase-1( prk-1) gene. The element is 3,298 bp long and flanked by a 200 bp direct repeat. 95 bp of the direct repeat are present in the coding region of prk-1. Rte-1 contains an open reading frame, in the opposite orientation of prk-1, potentially encoding 625 amino acids, with similarity to reverse transcriptases. The element is most similar to members of the non-LTR group of retrotransposable elements. There is weak homology of the predicted amino acid sequence of Rte-1 to several reverse transcriptase-like genes identified by the C. elegansgenome sequencing consortium, suggesting that there may be a large family of these elements. Southern blots indicate that there are approximately 10–15 additional Rte-1 elements in the C. elegansBristol N2 genome and a similar number is found in the genomes of two other geographically distinct strains. The insertion pattern of Rte-1 is polymorphic between these strains.

We have determined the solution structure of the DNA-binding domain of HIV-1 integrase by nuclear magnetic resonance spectroscopy. In solution, this carboxy-terminal region of integrase forms a homodimer, consisting of two structures that closely resemble Src-homology 3 (SH3) domains. Lys 264, previously identified by mutagenesis studies to be important for DNA binding of the integrase, as well as several adjacent basic amino acids are solvent exposed. The identification of an SH3-like domain in integrase provides a new potential target for drug design.

We have characterized a retrotransposon‐like element (Rte‐1) in C. elegans. It was identified while we were sequencing the pim related kinase‐1(prk‐1) gene. The element is 3,298 bp long and flanked by a 200 bp direct repeat. 95 bp of the direct repeat are present in the coding region of prk‐1. Rte‐1 contains an open reading frame, in the opposite orientation of prk‐1, potentially encoding 625 amino acids, with similarity to reverse transcriptases. The element is most similar to members of the non‐LTR group of retrotransposable elements. There is weak homology of the predicted amino acid sequence of Rte‐1 to several reverse transcriptase‐like genes identified by the C. elegansgenome sequencing consortium, suggesting that there may be a large family of these elements. Southern blots indicate that there are approximately 10–15 additional Rte‐1 elements in the C. elegansBristol N2 genome and a similar number is found in the genomes of two other geographically distinct strains. The insertion pattern of Rte‐1 is polymorphic between these strains.

Traditional reverse genetics on yeast, mice and other organisms uses homologous recombination with transgenic DNA to interrupt a target gene. Here we report that target-selected gene inactivation can be achieved in Caenorhabditis elegans with the use of chemical mutagens. We use PCR to selectively visualize deletions in genes of interest; the method is sensitive enough to permit detection of a single mutant among more than 15,000 wild types. A permanent frozen mutant collection of more than a million mutagenized animals has been established, and deletion mutants of several G-protein genes were isolated from it. The approach is suitable to be scaled up for systematic inactivation of all 17,000 C. elegans genes. Because it requires no transgenesis or cell culturing, it may also be applicable to small organisms usually considered to be outside the realm of reverse genetics (for example, other nematodes and insects). Any sequenced gene in any organism that can be handled in very large numbers can possibly be targeted in this way.

We have investigated the target choice of the related transposable elements Tc1 and Tc3 of the nematode C.elegans. The exact locations of 204 independent Tc1 insertions and 166 Tc3 insertions in an 1 kbp region of the genome were determined. There was no phenotyplc selection for the insertions. All insertions were into the sequence TA. Both elements have a strong preference for certain positions in the 1 kbp region. Hot sites for integration are not clustered or regularly spaced. The orientation of the integrated transposon has no effect on the distribution pattern. We tested several explanations for the target site preference. If simple structural features of the DNA (e.g. bends) would mark hot sites, we would expect the patterns of the two related transposons Tc1 and Tc3 to be similar; however we found them to be completely different. Furthermore we found that the sequence at the donor site has no effect on the choice of the new insertion site, because the insertion pattern of a transposon that jumps from a transgenlc donor site is identical to the insertion pattern of transposons jumping from endogenous genomic donor sites. The most likely explanation for the target choice is therefore that the primary sequence of the target site is recognized by the transposase. However, alignment of the Tc1 and Tc3 integration sites does not reveal a strong consensus sequence for either transposon.

Tc1 is a transposon present in several copies in the genome of all natural isolates of the nematode C.elegans; it is actively transposing in many strains. In those strains Tc1 insertion is the main cause of spontaneous mutations. The transposon contains one large ORF that we call TcA; we assume that the TcA protein is the transposase of id. We expressed TcA in E.coli, purified the protein and showed that it has a strong affinity for DNA (both single stranded and double stranded). A fusion protein of β-galactosidase and TcA also exhibits DNA binding; deletion derivatives of this fusion protein were tested for DNA binding. A deletion of 39 amino acids at the N-terminal region of TcA abolishes the DNA binding, whereas a deletion of 108 C-terminal amino acids does not affect DNA binding. This shows that the DNA binding domain of TcA is near the N-terminal region. The DNA binding capacity of TcA supports the assumption that TcA is a transposase of Tc1.

Before Integration of the human immunodeficiency virus (HIV) DNA, two nucleotides are removed from the 3′ ends of the viral DNA by the integrase (IN) protein. We studied the chemistry of this reaction, and found that IN mediates site-specific hydrolysis of a phosphodiester bond, resulting in release of a dinucleotide. A class of alcohols (including glycerol, 1,2-propanediol, but not 1,3-propanediol) can also act as nucleophile In this reaction, and likewise the alcoholic amino acids L-serine and L-threonine can be covalently linked to the dinucleotide. No evidence was found for a covalent linkage between the IN protein and this dinucleotide, suggesting that IN directs a single nucleophilic attack of water at the specific phosphodiester bond.

The integrase (IN) protein of the human Immunodeficiency virus (HIV) is required for specific cleavage of the viral DNA termini, and subsequent integration of the viral DNA Into target DNA. To identify the various domains of the IN protein we generated a series of IN deletion mutants as fusions to maltose-binding protein (MBP). The deletion mutants were tested for their ability to bind DNA, to mediate slte-speclflc cleavage of the viral DNA ends, and to carry out Integration and disintegration reactions. We found that the DNA-blndlng region resides between amlno acids 200 and 270 of the 288-residues HIV-1 IN protein. The catalytic domain of the protein was mapped between amlno acids 50 and 194. For the specific activities of IN, cleavage of the viral DNA and Integration, both the DNA-bindlng domain and the conserved amino-termlnal region of IN are required. These regions are dispensable however, for disintegration activity.

Integration of retroviral DNA into the host chromosome requires the integrase protein (IN). We overexpressed the IN proteins of human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) in E. coli and purified them. Both proteins were found to specifically cut two nucleotides off the ends of linear viral DNA, and to integrate viral DNA into target DNA. This demonstrates that HIV IN is the only protein required for integration of HIV DNA. Although the two types of IN proteins have only 53% amino acid sequence similarity, they act with equal efficiency on both type 1 and type 2 viral DNA. Binding of IN to DNA was tested: purified IN does not bind very specifically to viral DNA ends. Nevertheless, only viral DNA ends are cleaved and integrated. We interpret this as follows: in vitro quick aspecific binding to DNA is followed by slow specific cutting and integration. IN can not find viral DNA ends in the presence of an excess of aspecific DNA; in vivo this is not required since the IN protein is in constant proximity of viral DNA in the viral core particle.

The human immunodeficiency virus (HIV) integrase (IN) protein mediates an essential step in the retroviral lifecycle, the integration of viral DNA into human DNA. A DNA-binding domain of HIV IN has previously been identified in the C-terminal part of the protein. We tested truncated proteins of the C-terminal region of HIV-1 IN for DNA binding activity in two different assays: UV-crosslinking and southwestern blot analysis. We found that a polypeptide fragment of 50 amino acids (IN220−270) is sufficient for DNA binding. In contrast to full-length IN protein, this domain is soluble under low salt conditions. DNA binding of IN220−270 to both viral DNA and non-specific DNA occurs in an ion-independent fashion. Point mutations were introduced in 10 different amino acid residues of the DNA-binding domain of HIV-2 IN. Mutation of basic amino acid K264 results in strong reduction of DNA binding and of integrase activity.

The integrase (IN) protein of the human immunodeficiency virus (HIV) mediates two distinct reactions: (i) specific removal of two nucleotides from the 3′ ends of the viral DNA and (ii) integration of the viral DNA into target DNA. Although IN discriminates between specific (viral) DNA and nonspecific DNA in physical in vitro assays, a sequence-specific DNA-binding domain could not be identified in the protein. A nonspecific DNAbinding domain, however, was found at the C terminus of the protein. We examined the DNA-binding characteristics of HIV-1 IN, and found that a stable complex of IN and viral DNA is formed in the presence of Mn2$. The IN - viral DNA complex is resistant to challenge by an excess of competitor DNA. Stable binding of IN to the viral DNA requires that the protein contains an intact N-terminal domain and active site (in the central region of the protein), in addition to the C-terminal DNAbinding domain.