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Identification of novel non-autonomous CemaT transposable elements and evidence of their mobility within the C. elegans genome.

We describe here two new transposable elements, CemaT4 and CemaT5, that were identified within the sequenced genome of Caenorhabditis elegans using homology based searches. Five variants of CemaT4 were found, all non-autonomous and sharing 26 bp inverted terminal repeats (ITRs) and segments (152-367 bp) of sequence with similarity to the CemaT1 transposon of C. elegans. Sixteen copies of a short, 30 bp repetitive sequence, comprised entirely of an inverted repeat of the first 15 bp of CemaT4's ITR, were also found, each flanked by TA dinucleotide duplications, which are hallmarks of target site duplications of mariner-Tc transposon transpositions. The CemaT5 transposable element had no similarity to maT elements, except for sharing identical ITR sequences with CemaT3. We provide evidence that CemaT5 and CemaT3 are capable of excising from the C. elegans genome, despite neither transposon being capable of encoding a functional transposase enzyme. Presumably, these two transposons are cross-mobilised by an autonomous transposon that recognises their shared ITRs. The excisions of these and other non-autonomous elements may provide opportunities for abortive gap repair to create internal deletions and/or insert novel sequence within these transposons. The influence of non-autonomous element mobility and structural diversity on genome variation is discussed.

Animals↗

Assembly of the Tc1 and mariner transposition initiation complexes depends on the origins of their transposase DNA binding domains.

In this review, we focus on the assembly of DNA/protein complexes that trigger transposition in eukaryotic members of the IS630-Tc1-mariner (ITm) super-family, the Tc1- and mariner-like elements (TLEs and MLEs). Elements belonging to this super-family encode transposases with DNA binding domains of different origins, and recent data indicate that the chimerization of functional domains has been an important evolutionary aspect in the generation of new transposons within the ITm super-family. These data also reveal that the inverted terminal repeats (ITRs) at the ends of transposons contain three kinds of motif within their sequences. The first two are well known and correspond to the cleavage site on the outer ITR extremities, and the transposase DNA binding site. The organization of ITRs and of the transposase DNA binding domains implies that differing pathways are used by MLEs and TLEs to regulate transposition initiation. These differences imply that the ways ITRs are recognized also differ leading to the formation of differently organized synaptic complexes. The third kind of motif is the transposition enhancers, which have been found in almost all the functional MLEs and TLEs analyzed to date. Finally, in vitro and in vivo assays of various elements all suggest that the transposition initiation complex is not formed randomly, but involves a mechanism of oriented transposon scanning.

Amino Acid Sequence↗

Characterization of the genome structure of Bombyx mori densovirus (China isolate).

The genome of Bombyx mori densovirus (China isolate), termed as BmDNV-3, is composed of two kinds of different single-stranded linear DNA molecules (VD1 and VD2). In this study, the viral DNA molecules were purified and cloned into pUC119 vector, and the complete nucleotide sequence was determined. Sequence analysis showed that VD1 genome consisted of 6,543 nts including inverted terminal repeats (ITRs) of 224 nts, and VD2 genome consisted of 6,022 nts including ITRs of 524 nts. Comparison of the complete genome sequence between BmDNV-3 and BmDNV-2 (Yamanashi isolate) showed an identity of 98.4% in VD1 and 97.7% in VD2, with a total number of 228 bp substitutions, 11 bp deletions and 3 bp insertions found in BmDNV-3. A single nucleotide "A" deletion at nt 1589 in BmDNV-3 caused a frame shift mutation and brought about a premature stop codon, thus dividing VD2 of BmDNV-3 into two ORFs (named VD2 ORF1a and VD2 ORF1b) within that region, while there was only one ORF (named VD2 ORF1) in the corresponding region of BmDNV-2 (Yamanashi isolate). Comparative polymorphisms of ORFs and ITR regions of the two viral genomes showed that highly variable regions were mainly located in VD1 ORF3, VD1 ORF4, VD2 ORF2, and ITRs of BmDNV-3. Northern blots analysis revealed that VD1 had 1.1 kb and 1.5 kb transcripts from the left half of its plus strand, and one transcript about 3.3 kb from the right half of its minus strand. Sequencing of 3' and 5' RACE products showed that the 1.1 kb transcript started at nt 290 and ended at nt 1437, the 1.5 kb transcript started at nt 1423 and ended at nt 2931, and the 3.3 kb transcript started at nt 6287 and ended at nt 2922. These results help us to further understand the variation between different DNV genera and its possible causes, providing clues for studying the evolutionary history of densoviruses.

Amino Acid Sequence↗

Structure and reversion of an adenovirus type 2 mutant containing a duplication of the left end of the genome at the right end.

A mutant (dp 201) of human adenovirus type 2 has been isolated that has the left 3% (1110 bp) of the genome duplicated at the right terminus. The structure of the mutant genome was studied by restriction endonuclease analysis and by direct DNA sequence analysis. The duplication has resulted in a deletion of 10 bp of the right inverted terminal repeat (ITR) at the junction of right and left end sequences. This internal ITR is not active in initiation or termination of DNA synthesis. Two subclones of dp 201 that bear a deletion between map positions 2.1 and 6.8 encompassing early gene blocks E1a and E1b were also isolated. One of these isolates (dl 201.1) retains the left end duplication and the other isolate (dl 201.2) lacks the left end duplication and is an apparent revertant of dl 201.1. The original mutant dp 201 also reverts to a wild type sequence at a low rate. Since 10 base pairs of the right ITR are deleted in the mutant, reversion must involve a repair mechanism using one of the two complete left end ITRs in the mutant genome. The mechanisms by which this reversion may occur are discussed.

Adenoviruses, Human↗

Multiple reiteration of a 40-bp nucleotide sequence in the inverted terminal repeat of the genome of a canine adenovirus.

The DNA of a vaccine strain of canine adenovirus type 1 [ICHV vaccine; Connaught Laboratories, Ltd.; CAV-1(CLL)] has been cloned in plasmid pAT153 in the form of subgenomic BamHI digestion fragments. Analysis of the nucleotide sequences of cloned terminal fragments has revealed an inverted terminal repeat (ITR) with a minimum length of 198 nucleotides, including a tandem reiteration of the 40-bp nucleotide sequence from positions 14 to 53. The ITRs had the 5'-CATCATCAAT ... sequence typical of adenoviruses and the highly conserved sequence ATAATATAC (nucleotides 9-17) of human strains. Additionally, one BamHI A clone (left terminus) contained three sequential copies of the 40-bp sequence, and two BamHI C clones (right terminus) contained at least seven. These did not appear to be artifacts of cloning, since evidence was obtained that the multiple reiterations also occurred in DNA isolated from intact virus. By analogy with human adenoviruses, the repetitive sequence in the CAV-1(CLL) genome encompasses the entire nuclear factor I (NFI) binding site of the origin of DNA replication. Additionally, the 40-bp nucleotide sequence was found to contain the sequence AGG(N)4GCCTAA (nucleotides 27-39), which closely resembles the concensus sequence of the human adenovirus NFI binding site [TGG(N)6-7GCCAA; nucleotides 25-38]. It appears, therefore, that the Connaught CAV-1 vaccine contains reiterated copies of an essential part of the adenoviral origin of DNA replication. A mechanism is proposed for the generation of multiple reiterations of sequences in the right ITR, given an initial single tandem repeat in the left ITR.

Adenoviridae↗

Inverted terminal repeats and terminal proteins of the genomes of pneumococcal phages.

The nucleotide (nt) sequence at the ends of the genomes of the Streptococcus pneumoniae phages Cp-5 and Cp-7 has been determined and compared with the corresponding sequence of phage Cp-1. The genomes of phages Cp-5 and Cp-7 have inverted terminal repeats (ITRs) 343 and 347 bp long, respectively. In Cp-1 DNA the ITR is 236 bp long and the following 116 bp are 93% homologous. Some regions within the ITRs are conserved in the three genomes although the complete sequence of the ITRs is no more conserved than the rest of their genomes. The chromatographic behavior of their tryptic peptides suggests that the terminal proteins (TPs) of at least two of the phages are similar and that the TPs of the three pneumococcal phages differ markedly from that of the Bacillus subtilis phage psi 29.

Bacteriophages↗

A 16bp Rep binding element is sufficient for mediating Rep-dependent integration into AAVS1.

Adeno-associated virus (AAV) is a non-pathogenic virus and the only known eukaryotic virus capable of targeting human chromosome 19 for integration at a well-characterized AAVS1 site. Its site-specific integration is mediated by Rep68 and Rep78, viral proteins that bind to both the viral genome and AAVS1 site on ch19 through a specific Rep-binding element (RBE) located in both the viral genome and AAVS1. There are three RBEs in the AAV genome: two identical ones in both inverted terminal repeats (ITR) and another one in a recently discovered region termed the P5 integration efficiency element (P5IEE) that encompasses the viral P5 promoter. In order to identify the viral cis-acting sequence essential for Rep-mediated integration, we tested a series of constructs containing various lengths of P5IEE and compared the two RBEs from ITR (RBE(itr)) and P5IEE (RBE(p5)) in terms of their efficiency in Rep-dependent integration. Methods employed included a colony-forming assay, a PCR-based assay and Southern blotting analysis. We found that 16bp of the RBE cis-element was sufficient for mediating Rep-dependent site-specific integration. Furthermore, RBE(itr) was both more effective and specific than the RBE(p5) in Rep-dependent integration at the AAVS1 site. These findings added new information on the mechanism of Rep-dependent AAV genome insertion at the AAVS1 site and may be helpful in developing new high efficiency vectors for site-specific transgene integration.

Base Pairing↗

Enhanced transgene expression from single-stranded AAV vectors in human cells in vitro and in murine hepatocytes in vivo.

We identified that distal 10 nucleotides in the D-sequence in AAV2 inverted terminal repeat (ITR) share partial sequence homology to 1/2 binding site of glucocorticoid receptor-binding element (GRE). Here, we describe that (1) purified GR binds to AAV2 D-sequence, and the D-sequence competes with GR binding to its cognate binding site; (2) dexamethasone-mediated activation of GR pathway significantly increases the transduction efficiency of AAV2 vectors in human cells; (3) human osteosarcoma cells, U2OS, which lack expression of GR, are poorly transduced by AAV2 vectors, but stable transfection with a GR expression plasmid restores vector-mediated transgene expression; (4) replacement of the distal 10 nucleotides in the D-sequence of the AAV2 ITR with a full-length GRE consensus sequence significantly enhances transgene expression in human cells in vitro and in murine hepatocytes in vivo; and (5) none of the ITRs in AAV1, AAV3, AAV4, AAV5, and AAV6 genomes contains the GRE 1/2 binding site, and insertion of a full-length GRE consensus sequence in the AAV6-ITR also significantly enhances transgene expression from AAV6 vectors, both in vitro and in vivo. These novel vectors, termed generation Y AAV vectors, which are serotype, transgene, or promoter agnostic, should be useful in human gene therapy.

AAV vectors↗

Modulation of reflex and sleep related apnea by pedunculopontine tegmental and intertrigeminal neurons.

We describe and summarize here our recent findings about the role in respiration of two pontine structures that are not classically included in the pontine respiratory group: the pedunculopontine tegmental nucleus (PPT) and the intertrigeminal region (ITR). We also discuss significant contributions of other workers in the field, especially, S. Datta [Cell. Mol. Neurobiol. 17: 341-365, 1997], R. Lydic and H. Baghdoyan [Sleep, 25: 617-622, 2002], and N. Chamberlin and C. Saper [J. Neurosci. 18: 6048-6056, 1998], who postulated a role for the ITR in modulating reflex apnea. In anesthetized and freely moving rats we have consistently documented that PPT and ITR have a role in respiration. Neurochemical manipulations of each area affected the brainstem respiratory pattern generator and respiratory pattern variability,observed as spontaneous disturbances during sleep or as induced reflex apnea. Although the exact central mechanisms of apnea cannot be determined from our studies to date, we postulate that reflex and sleep-related apneas in rats share some common brainstem pathways, which may include PPT and ITR.

Animals↗

Promoter activity of left inverted terminal repeat and downstream sequences of porcine adenovirus type 3.

Early region 1 (E1) of porcine adenovirus type 3 (PAdV-3) consists of E1A and E1B transcription units. The authentic promoter region of E1A contains a TATA box at nucleotide position (nt) 449 and a bifunctional regulatory element between nt 374 and 431, which enhances the transcription of E1A, but represses that of E1B. Here, we investigated the role of the left inverted terminal repeat (ITR) and its downstream sequences (between nt 151 and 312) in the transcription of early viral genes, and viral replication. Mutant PAdV-3s without the authentic E1A promoter region could be rescued by transfection of mutant genomic DNA into fetal porcine retina cells. Moreover, the mutant PAdV-3s produced E1A-specific mRNA and remained viable in swine testis (ST) cells suggesting that the left-terminal 151 bp including the ITR, can serve as a promoter for E1A expression. However, mutant PAdV-3s containing deletion including authentic E1A promoter region, displayed both reduced steady-state levels of early gene mRNAs (E1A, E1B, E2A, E3, and E4) and decreased rate of viral replication in ST cells. Interestingly, mutant PAdV-3s containing the left-terminal 312 bp displayed increased transcription of early genes including E1A. Our results suggest that the left ITR of PAdV-3 contain the promoter like elements and the sequences (between nt 151 and 312) downstream of left ITR can enhance its promoter activity.

Adenovirus E1A Proteins↗

Structure-function analysis of the inverted terminal repeats of the sleeping beauty transposon.

Translocation of Sleeping Beauty (SB) transposon requires specific binding of SB transposase to inverted terminal repeats (ITRs) of about 230 bp at each end of the transposon, which is followed by a cut-and-paste transfer of the transposon into a target DNA sequence. The ITRs contain two imperfect direct repeats (DRs) of about 32 bp. The outer DRs are at the extreme ends of the transposon whereas the inner DRs are located inside the transposon, 165-166 bp from the outer DRs. Here we investigated the roles of the DR elements in transposition. Although there is a core transposase-binding sequence common to all of the DRs, additional adjacent sequences are required for transposition and these sequences vary in the different DRs. As a result, SB transposase binds less tightly to the outer DRs than to the inner DRs. Two DRs are required in each ITR for transposition but they are not interchangeable for efficient transposition. Each DR appears to have a distinctive role in transposition. The spacing and sequence between the DR elements in an ITR affect transposition rates, suggesting a constrained geometry is involved in the interactions of SB transposase molecules in order to achieve precise mobilization. Transposons are flanked by TA dinucleotide base-pairs that are important for excision; elimination of the TA motif on one side of the transposon significantly reduces transposition while loss of TAs on both flanks of the transposon abolishes transposition. These findings have led to the construction of a more advanced transposon that should be useful in gene transfer and insertional mutagenesis in vertebrates.

Base Sequence↗

Subdivisions of inferior temporal cortex in squirrel monkeys make dissociable contributions to visual learning and memory.

Inferior temporal cortex of squirrel monkeys consists of caudal (ITC), intermediate (ITI), and rostral (ITR) subdivisions, possibly homologous to TEO, posterior TE, and anterior TE of macaque monkeys. The present study compared visual learning in squirrel monkeys with ablations of ITC; ITI and ITR (group ITRd); or ITI, ITR, and more ventral cortex, including perirhinal cortex (group ITR+), with visual learning in unoperated controls. The ITC monkeys had significant impairments on pattern discriminations and milder deficits on delayed non-matching to sample (DNMS) of objects. The ITRd monkeys had deficits on some pattern discriminations but not on DNMS. The ITRd monkeys were significantly impaired on DNMS and some pattern discriminations. These results are similar to those found in macaques and support the proposed homologies.

Analysis of Variance↗

Targeted transgene integration into transgenic mouse fibroblasts carrying the full-length human AAVS1 locus mediated by HSV/AAV rep(+) hybrid amplicon vector.

Herpes simplex virus type 1/adeno-associated virus (HSV/AAV) rep(+) hybrid amplicon vectors containing AAV inverted terminal repeats (ITRs) and rep gene sequences can mediate site-specific integration into the human genome. In this study, we have generated and characterized the first transgenic mice that bear the full-length (8.2 kb) human AAVS1 locus. Immortalized mouse embryonic fibroblasts from this mouse line were transduced with the rep(+), rep(-) (containing only ITRs flanking the transgene) hybrid amplicon vectors, and the standard amplicon vector to determine stable integration frequency and the site of integration. Transduction of transgenic fibroblasts resulted in a 10-fold higher stable integration frequency with rep(+) hybrid amplicon vector than with rep(-) or standard amplicon vectors. Southern blot analysis of genomic DNA from transgenic cells stably transduced with the rep(+) hybrid amplicon vector revealed site-specific integration of transgenes at the AAVS1 locus in 50% of clones. Some site-specific and random integration events were limited to the ITR-flanked transgene cassette. In contrast, transduction of transgenic mouse cells with the rep(-) or standard amplicon vectors resulted in random integrations of the entire rep(-) hybrid amplicon or amplicon DNA that were incorporated into the host genome as a concatenate of various sizes. These results demonstrate for the first time that the genome of transgenic mice bearing the human AAVS1 locus serves as a platform for site-specific integration of AAV ITR-flanked transgene cassettes within the hybrid amplicon vector in the presence of Rep.

Animals↗

A genetic screen identifies a cellular regulator of adeno-associated virus.

Adeno-associated virus type 2 (AAV2) is a human parvovirus that has attracted attention as a vector for gene transfer. Replication and site-specific integration of the wild-type virus requires binding of the AAV2 Rep proteins to a cis-regulatory element named the Rep recognition sequence (RRS). RRS motifs are found within the cellular AAVS1 integration locus, the viral p5 promoter, and the inverted terminal repeats (ITRs). Here we report the design of a genetic screen based on the yeast one-hybrid assay to identify cellular RRS-binding proteins. We show that the human zinc finger 5 protein (ZF5) binds specifically to RRS motifs in vitro and in vivo. ZF5 is a highly conserved and ubiquitously expressed transcription factor that contains five C-terminal zinc fingers and an N-terminal POZ domain. Ectopic expression of ZF5 leads to an ITR-dependent repression of the autologous p5 promoter and reduces both AAV2 replication and the production of recombinant AAV2. By using deletion and substitution mutants we show that two different domains of ZF5 contribute to AAV2 repression. Negative regulation of the p5 promoter requires the POZ domain, whereas viral replication is inhibited by the zinc finger domain, likely by competing with Rep for binding to the ITR. Identification and characterization of proteins that bind the ITR, the only viral genetic element retained in AAV2 vectors, will lead to new insights into the unique life cycle of AAV2 and will suggest improvements important for its application as a gene therapy vector.

Base Sequence↗

Spontaneous deletions and duplications of sequences in the genome of cowpox virus.

Examination of the genomes of 10 white-pock variants of cowpox virus strain Brighton red (CPV-BR) revealed that 9 of them had lost 32 to 38 kilobase pairs (kbp) from their right-hand ends and that the deleted sequences had been replaced by inverted copies of regions from 21 to 50 kbp long from the left-hand end of the genome. These variants thus possess inverted terminal repeats (ITRs) from 21 to 50 kbp long; all are longer than the ITRs of CPV-BR (10 kbp). The 10th variant is a simple deletion mutant that has lost the sequences between 32 and 12 kbp from the right-hand end of the genome. The limits of the inner ends of the observed deletions (between 32 and 38 kbp from the right-hand end of the CPV-BR genome) appear to be defined by the location of the nearest essential gene on the one hand and the location of the gene that encodes "pock redness" on the other. The genomes of the deletion/duplication white-pock variants appear to have been generated either by single crossover recombinational events between two CPV-BR genomes aligned in opposite directions or by the nonreciprocal transfer of genetic information. The sites where such recombination/transfer occurred were sequenced in four variants. In all of them, the sequences adjacent to such sites show no sequence homology or any other unusual structural feature. The analogous sites at the internal ends of the two ITRs of CPV-BR also were sequenced and also show no unusual features. It is likely that the ITRs of CPV-BR and of its white-pock variants, and probably those of other orthopox-virus genomes, arise as a result of nonhomologous recombination or by random nonreciprocal transfer of genetic information.

Base Sequence↗

Terminal region recognition factor 1, a DNA-binding protein recognizing the inverted terminal repeats of the pGKl linear DNA plasmids.

The yeast linear DNA plasmids pGKl1 and pGKl2 contain inverted terminal repeats (ITRs) and terminal proteins covalently bound to the 5' termini of each plasmid. The presence of these features suggests a protein-primed mechanism of DNA replication, similar to that exemplified by mammalian adenovirus and phi 29 phage of Bacillus subtilis. In this paper, we report the identification of an activity in cytoplasmic extracts of yeast harboring the pGKl plasmids that recognizes the termini of both pGKl1 and pGKl2. We call this activity TRF1, for terminal region recognition factor 1. Deletion analyses and DNase I protection experiments demonstrate that the activity recognizes base pairs 107-183 within the ITR of pGKl1, and base pairs 126-179 within the ITR of pGKl2. The presence of T-tracts within these two regions, but otherwise dissimilar nucleotide sequences, suggests that TRF1 recognizes a common structural feature within the ITRs of the two plasmids. TRF1 has been partially purified from yeast cytoplasmic extracts and Southwestern analysis indicates that the apparent molecular mass of the protein is 16 kDa. By expressing three open reading frames from pGKl2 in Escherichia coli, we found that open reading frame 10 (ORF10) of pGKl2 encodes TRF1. The sequence of the ORF10 gene product indicates that TRF1 is a highly basic protein of small molecular mass. Comparison of TRF1 with other DNA-binding proteins known to recognize the terminal regions of linear DNAs, such as NFI and NFIII involved in adenovirus DNA replication, and phi 29 p6, involved in phi 29 DNA replication, indicates that TRF1 has a different mode of binding.

Amino Acid Sequence↗

Oscillatory Ca2+ signaling in the isolated Caenorhabditis elegans intestine: role of the inositol-1,4,5-trisphosphate receptor and phospholipases C beta and gamma.

Defecation in the nematode Caenorhabditis elegans is a readily observable ultradian behavioral rhythm that occurs once every 45-50 s and is mediated in part by posterior body wall muscle contraction (pBoc). pBoc is not regulated by neural input but instead is likely controlled by rhythmic Ca(2+) oscillations in the intestinal epithelium. We developed an isolated nematode intestine preparation that allows combined physiological, genetic, and molecular characterization of oscillatory Ca(2+) signaling. Isolated intestines loaded with fluo-4 AM exhibit spontaneous rhythmic Ca(2+) oscillations with a period of approximately 50 s. Oscillations were only detected in the apical cell pole of the intestinal epithelium and occur as a posterior-to-anterior moving intercellular Ca(2+) wave. Loss-of-function mutations in the inositol-1,4,5-trisphosphate (IP(3)) receptor ITR-1 reduce pBoc and Ca(2+) oscillation frequency and intercellular Ca(2+) wave velocity. In contrast, gain-of-function mutations in the IP(3) binding and regulatory domains of ITR-1 have no effect on pBoc or Ca(2+) oscillation frequency but dramatically increase the speed of the intercellular Ca(2+) wave. Systemic RNA interference (RNAi) screening of the six C. elegans phospholipase C (PLC)-encoding genes demonstrated that pBoc and Ca(2+) oscillations require the combined function of PLC-gamma and PLC-beta homologues. Disruption of PLC-gamma and PLC-beta activity by mutation or RNAi induced arrhythmia in pBoc and intestinal Ca(2+) oscillations. The function of the two enzymes is additive. Epistasis analysis suggests that PLC-gamma functions primarily to generate IP(3) that controls ITR-1 activity. In contrast, IP(3) generated by PLC-beta appears to play little or no direct role in ITR-1 regulation. PLC-beta may function instead to control PIP(2) levels and/or G protein signaling events. Our findings provide new insights into intestinal cell Ca(2+) signaling mechanisms and establish C. elegans as a powerful model system for defining the gene networks and molecular mechanisms that underlie the generation and regulation of Ca(2+) oscillations and intercellular Ca(2+) waves in nonexcitable cells.

Animals↗

Inositol 1,4,5-trisphosphate signaling regulates mating behavior in Caenorhabditis elegans males.

Complex behavior requires the coordinated action of the nervous system and nonneuronal targets. Male mating in Caenorhabditis elegans consists of a series of defined behavioral steps that lead to the physiological outcomes required for successful impregnation. We demonstrate that signaling mediated by inositol 1,4,5-trisphosphate (IP(3)) is required at several points during mating. Disruption of IP(3) receptor (itr-1) function results in dramatic loss of male fertility, due to defects in turning behavior (during vulva location), spicule insertion and sperm transfer. To elucidate the signaling pathways responsible, we knocked down the six C. elegans genes encoding phospholipase C (PLC) family members. egl-8, which encodes PLC-beta, functions in spicule insertion and sperm transfer. itr-1 and egl-8 are widely expressed in the male reproductive system. An itr-1 gain-of-function mutation rescues infertility caused by egl-8 RNA interference, indicating that egl-8 and itr-1 function together as central components of the signaling events controlling sperm transfer.

Animals↗