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Fertilization is the process that leads to the formation of a diploid zygote from two haploid gametes. This is achieved through a complex series of cell-to-cell interactions between a sperm and an egg. The final event of fertilization is the fusion of the gametes' membranes, which allows the delivery of the sperm genetic material into the egg cytoplasm. In vivo studies in the laboratory mouse have led to the discovery of membrane proteins that are essential for the fusion process in both the sperm and egg. Specifically, the sperm protein Izumo1 was shown to be necessary for normal fertility. Izumo1-deficient spermatozoa fail to fuse with the egg plasma membrane. Izumo1 is a member of the Immunoglobulin Superfamily of proteins, which are known to be involved in cell adhesion. Here, we describe BART97b, a new mouse line with a recessive mutation that displays a fertilization block associated with a failure of sperm fusion. BART97b mutants carry a deletion that inactivates Spaca6, a previously uncharacterized gene expressed in testis. Similar to Izumo1, Spaca6 encodes an immunoglobulin-like protein. We propose that the Spaca6 gene product may, together with Izumo1, mediate sperm fusion by binding an as yet unidentified egg membrane receptor.

Lens induction is a classical embryologic model to study cell fate determination. It has been proposed earlier that specific changes in core histone modifications accompany the process of cell fate specification and determination. The lysine acetyltransferases CBP and p300 function as principal enzymes that modify core histones to facilitate specific gene expression. Herein, we performed conditional inactivation of both CBP and p300 in the ectodermal cells that give rise to the lens placode. Inactivation of both CBP and p300 resulted in the dramatic discontinuation of all aspects of lens specification and organogenesis, resulting in aphakia. The CBP/p300(-/-) ectodermal cells are viable and not prone to apoptosis. These cells showed reduced expression of Six3 and Sox2, while expression of Pax6 was not upregulated, indicating discontinuation of lens induction. Consequently, expression of αB- and αA-crystallins was not initiated. Mutant ectoderm exhibited markedly reduced levels of histone H3 K18 and K27 acetylation, subtly increased H3 K27me3 and unaltered overall levels of H3 K9ac and H3 K4me3. Our data demonstrate that CBP and p300 are required to establish lens cell-type identity during lens induction, and suggest that posttranslational histone modifications are integral to normal cell fate determination in the mammalian lens.

Correct development of the cerebellum requires coordinated sonic hedgehog (Shh) signaling from Purkinje to granule cells. How Shh expression is regulated in Purkinje cells is poorly understood. Using a novel tyrosinase minigene-tagged Sleeping Beauty transposon-mediated mutagenesis, which allows for coat color-based genotyping, we created mice in which the Ski/Sno family transcriptional co-repressor 2 (Skor2) gene is deleted. Loss of Skor2 leads to defective Purkinje cell development, a severe reduction of granule cell proliferation and a malformed cerebellum. Skor2 is specifically expressed in Purkinje cells in the brain, where it is required for proper expression of Shh. Skor2 overexpression suppresses BMP signaling in an HDAC-dependent manner and stimulates Shh promoter activity, suggesting that Skor2 represses BMP signaling to activate Shh expression. Our study identifies an essential function for Skor2 as a novel transcriptional regulator in Purkinje cells that acts upstream of Shh during cerebellum development.

Autism spectrum disorder (ASD) diagnoses are behaviorally based with no defined universal biomarkers, occur at a 1:110 ratio in the population, and predominantly affect males compared to females at approximately a 4:1 ratio. One approach to investigate and identify causes of ASD is to use organisms that display abnormal behavioral responses that model ASD-related impairments. This study describes a novel transgenic mouse, MALTT, which was generated using a forward genetics approach. It was determined that the transgene integrated within a non-coding region on the X chromosome. The MALTT line exhibited a complete repertoire of ASD-like behavioral deficits in all three domains required for an ASD diagnosis: reciprocal social interaction, communication, and repetitive or inflexible behaviors. Specifically, MALTT male mice showed deficits in social interaction and interest, abnormalities in pup and juvenile ultrasonic vocalization communications, and exhibited a repetitive stereotypy. Abnormalities were also observed in the domain of sensory function, a secondary phenotype prevalently associated with ASD. Mapping and expression studies suggested that the Fam46 gene family may be linked to the observed ASD-related behaviors. The MALTT line provides a unique genetic model for examining the underlying biological mechanisms involved in ASD-related behaviors.

The mitochondrion is involved in energy generation, apoptosis regulation, and calcium homeostasis. Mutations in genes involved in mitochondrial processes often result in a severe phenotype or embryonic lethality, making the study of mitochondrial involvement in aging, neurodegeneration, or reproduction challenging. Using a transgenic insertional mutagenesis strategy, we generated a mouse mutant, Immp2lTg(Tyr)979Ove, with a mutation in the inner mitochondrial membrane peptidase 2-like (Immp2l) gene. The mutation affected the signal peptide sequence processing of mitochondrial proteins cytochrome c1 and glycerol phosphate dehydrogenase 2. The inefficient processing of mitochondrial membrane proteins perturbed mitochondrial function so that mitochondria from mutant mice manifested hyperpolarization, higher than normal superoxide ion generation, and higher levels of ATP. Homozygous Immp2lTg(Tyr)979Ove females were infertile due to defects in folliculogenesis and ovulation, whereas mutant males were severely subfertile due to erectile dysfunction. The data suggest that the high superoxide ion levels lead to a decrease in the bioavailability of nitric oxide and an increase in reactive oxygen species stress, which underlies these reproductive defects. The results provide a novel link between mitochondrial dysfunction and infertility and suggest that superoxide ion targeting agents may prove useful for treating infertility in a subpopulation of infertile patients.

A significant barrier to exploiting the full potential of the rat as a biomedical model is the lack of tools to easily modify its germline. Here we show that a tyrosinase-tagged Sleeping Beauty transposon can be used as a simple, efficient method to generate rat mutants in vivo. By making two lines of transgenic rats, one carrying the transposon and another expressing the transposase in germ cells, we are able to obtain bigenic males in which transposition occurs in the germ cells. We show that transposition leads to the appearance of new coat colors in the offspring. Using such bigenic males, we obtained an average of 1.2 transpositions per gamete and identified 19 intragenic integration events among 96 transposition sites that were sequenced. In addition, gene trapping was confirmed and rats with evidence for transposon-induced dominant ocular anomalies were identified. These data suggest that the modified Sleeping Beauty transposon represents a powerful new tool for producing molecularly defined mutagenesis in the rat.

The transgenic mouse line OVE459 carries a transgene-induced insertional mutation resulting in autosomal recessive congenital hydrocephalus. Homozygous transgenic animals experience ventricular dilation with perinatal onset and are noticeably smaller than hemizygous or non-transgenic littermates within a few days after birth. Fluorescence in situ hybridization (FISH) revealed that the transgene inserted in a single locus on mouse Chromosome (chr) 8, region D2-E1. Genetic crosses between hemizygous OVE459 mice and mice heterozygous for the spontaneous mutation hydrocephalus-3 (hy3) produced hydrocephalic offspring with a frequency of 22%, demonstrating that these two mutations are allelic. A genomic library was made by using DNA from homozygous OVE459 mice, and genomic DNA flanking the transgene insertion site was isolated and sequenced. A PCR polymorphism between C57BL/6 DNA and Mus spretus was used to map the location of the transgene insert to 1.06 cM +/- 0.75 proximal to D8Mit152 by using the Jackson Laboratory Backcross DNA Panel Mapping Resource. Furthermore, sequence analysis from a mouse bacterial artificial chromosome (BAC) clone, positive for unique markers on both sides of the transgene insertion site, demonstrated that the genomic DNAs flanking each side of the transgene insertion are physically separated by approximately 51 kb on the wild-type mouse chromosome.

7 p.- 2 fig.-- et al., Premature ovarian failure is a major cause of female infertility. The genetic causes of this disorder remain unknown in most patients. Using whole-exome sequence analysis of a large consanguineous family with inherited premature ovarian failure, we identified a homozygous 1-bp deletion inducing a frameshift mutation in STAG3 on chromosome 7. STAG3 encodes a meiosis-specific subunit of the cohesin ring, which ensures correct sister chromatid cohesion. Female mice devoid of Stag3 are sterile, and their fetal oocytes are arrested at early prophase I, leading to oocyte depletion at 1 week of age., Supported by grants from the University Paris Diderot-Paris 7(to Drs. Caburet, Veitia, and Fellous), the Ligue Nationale contre le Cancer and the Centre National de la Recherche Scientifique (to Drs. Caburet and Veitia), the GIS (Groupement d’Intérêt Scientifique) Institut des Maladies Rares (to Drs. Caburet and Fellous), the Doris Duke Charitable Foundation and National Institute of Child Health and Human Development (NICHD) (RO1HD068138 and R01HD044513, to Drs. Arboleda and Vilain), the NICHD (F31HD068136) and the UCLA Medical Scientist Training Program (both to Dr. Arboleda), and the Ministerio de Economía y Competitividad (SAF2011-25252) and Junta de Castilla y León (both to Drs. Llano and Pendás). The lentiviral mutagenesis studies were supported by the Ocular Development Fund (to Dr. Overbeek).

The Notch signaling pathway is thought to regulate multiple stages of inner ear development. Mutations in the Notch signaling pathway cause disruptions in the number and arrangement of hair cells and supporting cells in sensory regions of the ear. In this study we identify an insertional mutation in the mouse Sfswap gene, a putative splicing factor, that results in mice with vestibular and cochlear defects that are consistent with disrupted Notch signaling. Homozygous Sfswap mutants display hyperactivity and circling behavior consistent with vestibular defects, and significantly impaired hearing. The cochlea of newborn Sfswap mutant mice shows a significant reduction in outer hair cells and supporting cells and ectopic inner hair cells. This phenotype most closely resembles that seen in hypomorphic alleles of the Notch ligand Jagged1 (Jag1). We show that Jag1; Sfswap compound mutants have inner ear defects that are more severe than expected from simple additive effects of the single mutants, indicating a genetic interaction between Sfswap and Jag1. In addition, expression of genes involved in Notch signaling in the inner ear are reduced in Sfswap mutants. There is increased interest in how splicing affects inner ear development and function. Our work is one of the first studies to suggest that a putative splicing factor has specific effects on Notch signaling pathway members and inner ear development., Author Summary The organ of Corti is a sensory structure in the cochlea that mediates our sense of hearing. It consists of one row of inner hair cells and three rows of outer hair cells, together with an array of neighboring supporting cells. The precise arrangement of these different cell types is regulated very tightly by a number of signaling pathways during embryonic development, and mutations in genes that regulate this pattern often lead to deafness. We have generated a mouse mutant containing a lentiviral insertion in a gene encoding a putative RNA splicing factor called Sfswap. Homozygous mutant mice have hearing and balance defects, and have an abnormal arrangement of hair cells in their cochlea. These defects are consistent with defects in the Notch signaling pathway. We show that Sfswap mutants interact genetically with a mutation in Jagged1, which encodes a Notch ligand. We show that expression of some genes involved in Notch signaling is disrupted in Sfswap mutant mice. Our work is one of the first studies to show that a putative splicing factor has specific effects on Notch signaling pathway members and on inner ear development.

A distinctive feature of gonadal maturation in mammals is the movement to an extraabdominal location. Testicular descent is a complex, multistage process whereby the embryonic gonads migrate from their initial abdominal position to the scrotum. Failure in this process results in cryptorchidism, a frequent congenital birth defect in humans. We report here a new mouse transgenic insertional mutation, cryptorchidism with white spotting (crsp). Males homozygous for crsp exhibit a high intraabdominal position of the testes, associated with complete sterility. Heterozygous males have a wild-type phenotype, and homozygous females are fertile. Surgically descended testes in crsp/crsp males show normal spermatogenesis. Using FISH and genetic analyses, the transgenic insert causing the crsp mutation has been mapped to the distal part of mouse chromosome 5. Transgene integration resulted in a 550-kb deletion located upstream of the Brca2 gene. A candidate gene encoding a novel G protein-coupled receptor (Great) with an expression pattern suggesting involvement in testicular descent has been identified.

In most mammals, male development is triggered by the transient expression of the Y-chromosome gene, Sry, which initiates a cascade of gene interactions ultimately leading to the formation of a testis from the indifferent fetal gonad. Several genes, in particular Sox9, have a crucial role in this pathway. Despite this, the direct downstream targets of Sry and the nature of the pathway itself remain to be clearly established. We report here a new dominant insertional mutation, Odsex (Ods), in which XX mice carrying a 150-kb deletion (approximately 1 Mb upstream of Sox9) develop as sterile XX males lacking Sry. During embryogenesis, wild-type XX fetal gonads downregulate Sox9 expression, whereas XY and XX Ods/+ fetal gonads upregulate and maintain its expression. We propose that Ods has removed a long-range, gonad-specific regulatory element that mediates the repression of Sox9 expression in XX fetal gonads. This repression would normally be antagonized by Sry protein in XY embryos. Our data are consistent with Sox9 being a direct downstream target of Sry and provide genetic evidence to support a general repressor model of sex determination in mammals.

Bent tail (BN:) is a spontaneous, semi-dominant mutation on the mouse X chromosome that produces tail deformities and, rarely, open neural tube defects. Analysis of 292 normal male and affected male and female progeny from an intraspecific back-cross involving BN: supports a gene order of cen-DXMit89-18.5 +/- 2.3 cM-DXMit166-1.4 +/- 0.7 cM-BN:-1.0 +/- 0.6 cM-DXMit140 -4.8 +/- 1.3 cM-DXBay6-tel. A high frequency of sex chromosomal non-disjunction, unrelated to the BN: mutation, was also identified in the background strain. Refined genetic and physical mapping of the BN: critical region demonstrate that the mutation is associated with a

Mice homozygous for the transgenic insertion in line OVE250 exhibit severe progressive glomerulonephritis. Ultrastructural changes in the glomerular basement membrane (GBM) at 2 weeks of age resemble those in Alport syndrome. The transgenic insertion site was mapped by FISH to mouse chromosome 1 close to Pax3. Genetic and molecular analyses identified a deletion of genomic DNA at the transgene insertion site. Exons 1 through 12 of the collagen IV gene Col4a4, exons 1 and 2 of the adjacent Col4a3 gene, and the intergenic promoter region are deleted. Transcripts of Col4a3 and Col4a4 are undetectable in mutant kidney, and both proteins are missing from the GBM. Persistent cellular proliferation in mutant kidneys suggests that interaction with the extracellular matrix may be important for cell maturation. Evolutionarily conserved sequence elements in the promoter regions of human and mouse Col4a3 and Col4a4 include a 19-bp element that was tandemly duplicated in the human lineage and a CTC box element common to several genes encoding extracellular matrix proteins. This new animal model of Alport syndrome, Col4Delta3-4, lacks both alpha3 and alpha4 chains of collagen IV and exhibits an earlier disease onset than mice lacking alpha3 only.

Carnitine palmitoyltransferase (CPT)-I catalyses the transfer of long-chain fatty acids from CoA to carnitine for translocation across the mitochondrial inner membrane. Expression of the 'liver' isoform of the CPT-I gene (CPT-Ialpha) is subject to developmental, hormonal and tissue-specific regulation. To understand the basis for control of CPT-Ialpha gene expression, we have characterized the proximal promoter of the CPT-Ialpha gene. Here, we report the sequence of 6839 base pairs of the promoter and the localization of the rat CPT-Ialpha gene to region q43 on chromosome 1. Our studies show that the first 200 base pairs of the promoter are sufficient to drive transcription of the CPT-Ialpha gene. Within this region are two sites that bind both Sp1 and Sp3 transcription factors. In addition, nuclear factor Y (NF-Y) binds the proximal promoter. Mutation at the Sp1 or NF-Y sites severely decreases transcription from the CPT-Ialpha promoter. Other protein binding sites were identified within the first 200 base pairs of the promoter by DNase I footprinting, and these elements contribute to CPT-Ialpha gene expression. Our studies demonstrate that CPT-Ialpha is a TATA-less gene which utilizes NF-Y and Sp proteins to drive basal expression.

Two frequent protein variants of glutamate pyruvate transaminase (GPT) (E.C.2.6.1.2) have been used as genetic markers in humans for more than two decades, although chromosomal mapping of the GPT locus in the 1980s produced conflicting results. To resolve this conflict and develop useful DNA markers for this gene, we isolated and characterized cDNA and genomic clones of GPT. We have definitively mapped human GPT to the terminus of 8q using several methods. First, two cosmids shown to contain the GPT sequence were derived from a chromosome 8-specific library. Second, by fluorescencein situhybridization, we mapped the cosmid containing the human GPT gene to chromosome band 8q24.3. Third, we mapped the ratgptcDNA to the syntenic region of rat chromosome 7. Finally, PCR primers specific to human GPT amplify sequences contained within a “half-YAC” from the long arm of chromosome 8, that is, a YAC containing the 8q telomere. The human GPT genomic sequence spans 2.7 kb and consists of 11 exons, ranging in size from 79 to 243 bp. The exonic sequence encodes a protein of 495 amino acids that is nearly identical to the previously reported protein sequence of human GPT-1. The two polymorphic GPT isozymes are the result of a nucleotide substitution in codon 14, coding for a histidine in GPT-1 and an asparagine in GPT-2, which causes a gain or loss of anNlaIII restriction site. In addition, a cosmid containing the GPT sequence also contains a previously unmapped, polymorphic microsatellite sequence, D8S421. The cloned GPT gene and associated polymorphisms will be useful for linkage and physical mapping of disease loci that map to the terminus of 8q, including atypical vitelliform macular dystrophy (VMD1) and epidermolysis bullosa simplex, type Ogna (EBS1). In addition, this will be a useful system for characterizing the telomeric region of 8q. Finally, determination of the molecular basis of the GPT isozyme variants will permit PCR-based detection of this worldwide polymorphism.

Congenital ichthyoses are life-threatening conditions in humans. We describe here the identification and molecular characterization of a novel recessive mutation in mice that results in newborn lethality with severe congenital lamellar ichthyosis. Mutant newborns have a taut, shiny, non-expandable epidermis that resembles cornified manifestations of autosomal-recessive congenital ichthyosis in humans. The skin is stretched so tightly that the newborn mice are immobilized. The genetic defect was mapped to a region near the proximal end of chromosome 2 by SNP analysis, suggesting Fatp4/Slc27a4 as a candidate gene. FATP4 mutations in humans cause ichthyosis prematurity syndrome (IPS), and mutations of Fatp4 in mice have previously been found to cause a phenotype that resembles human congenital ichthyoses. Characterization of the Fatp4 cDNA revealed a fusion of exon 8 to exon 10, with deletion of exon 9. Genomic sequencing identified an A to T mutation in the splice donor sequence at the 3'-end of exon 9. Loss of exon 9 results in a frame shift mutation upstream from the conserved very long-chain acyl-CoA synthase (VLACS) domain. Histological studies revealed that the mutant mice have defects in keratinocyte differentiation, along with hyperproliferation of the stratum basale of the epidermis, a hyperkeratotic stratum corneum, and reduced numbers of secondary hair follicles. Since Fatp4 protein is present primarily at the stratum granulosum and the stratum spinosum, the hyperproliferation and the alterations in hair follicle induction suggest that very long chain fatty acids, in addition to being required for normal cornification, may influence signals from the stratum corneum to the basal cells that help to orchestrate normal skin differentiation.

Pseudoachondroplasia (PSACH) is a well characterized dwarfing condition mapping to chromosome 19p12-13.1. Cartilage oligomeric matrix protein (COMP), a cartilage specific protein, maps to the same location within a contig that spans the PSACH locus. Using single strand conformation polymorphism (SSCP) analysis and nucleotide sequencing we have identified COMP mutations in eight familial and isolated PSACH cases. All mutations involve either a single base-pair change or a three base-pair deletion in exon 17B. Six mutations delete or change a well conserved aspartic acid residue within the calcium-binding type 3 repeats. These results demonstrate that mutations in the COMP gene cause pseudochondroplasia.

We report on an infant who presented at birth with some characteristics of trisomy 18 syndrome, including low birth weight, facial abnormalities, overlapping fingers, and congenital heart defects. On chromosome analysis, no additional chromosome 18 was observed and both chromosome 18 homologues appeared normal. However, a small piece of chromosomal material of unknown origin was detected at the tip of the long arm of chromosome 1. Fluorescence in situ hybridization (FISH) using whole chromosome 18 painting probes disclosed no additional hybridization at the telomere of 1q, suggesting that the material was derived from another chromosome. Further chromosome painting experiments suggested that the telomeric addition was of chromosome 1 origin. To identify subchromosomal regions involved in the rearrangement, additional FISH analyses were performed using single copy and repetitive DNA probes mapping to different portions of chromosome 1. The analyses showed that probes mapping to 1q34-43 were duplicated in the derivative chromosome 1. In addition, a DNA probe mapping to 1q44 was found to be deleted from the derivative chromosome 1. Our composite analysis suggests that a deletion and a duplication of chromosome 1q can result in some of the clinical findings usually associated with trisomy 18 syndrome. These results demonstrate the usefulness of FISH analysis when karyotype analysis is not consistent with the clinical description.

The head bobber transgenic mouse line, produced by pronuclear integration, exhibits repetitive head tilting, circling behavior, and severe hearing loss. Transmitted as an autosomal recessive trait, the homozygote has vestibular and cochlea inner ear defects. The space between the semicircular canals is enclosed within the otic capsule creating a vacuous chamber with remnants of the semicircular canals, associated cristae, and vestibular organs. A poorly developed stria vascularis and endolymphatic duct is likely the cause for Reissner’s membrane to collapse post-natally onto the organ of Corti in the cochlea. Molecular analyses identified a single integration of ~3 tandemly repeated copies of the transgene, a short duplicated segment of chromosome X and a 648 kb deletion of chromosome 7(F3). The three known genes (Gpr26, Cpxm2, and Chst15) in the deleted region are conserved in mammals and expressed in the wild-type inner ear during vestibular and cochlea development but are absent in homozygous mutant ears. We propose that genes critical for inner ear patterning and differentiation are lost at the head bobber locus and are candidate genes for human deafness and vestibular disorders.

The mitochondrion is involved in energy generation, apoptosis regulation, and calcium homeostasis. Mutations in genes involved in mitochondrial processes often result in a severe phenotype or embryonic lethality, making the study of mitochondrial involvement in aging, neurodegeneration, or reproduction challenging. Using a transgenic insertional mutagenesis strategy, we generated a mouse mutant, Immp2lTg(Tyr)979Ove, with a mutation in the inner mitochondrial membrane peptidase 2-like (Immp2l) gene. The mutation affected the signal peptide sequence processing of mitochondrial proteins cytochrome c1 and glycerol phosphate dehydrogenase 2. The inefficient processing of mitochondrial membrane proteins perturbed mitochondrial function so that mitochondria from mutant mice manifested hyperpolarization, higher than normal superoxide ion generation, and higher levels of ATP. Homozygous Immp2lTg(Tyr)979Ove females were infertile due to defects in folliculogenesis and ovulation, whereas mutant males were severely subfertile due to erectile dysfunction. The data suggest that the high superoxide ion levels lead to a decrease in the bioavailability of nitric oxide and an increase in reactive oxygen species stress, which underlies these reproductive defects. The results provide a novel link between mitochondrial dysfunction and infertility and suggest that superoxide ion targeting agents may prove useful for treating infertility in a subpopulation of infertile patients.

In mouse, the Sxr(b) deletion interval (delta Sxr(b)) maps to the small short arm of the Y chromosome and is known to contain gene(s) required for normal spermatogenesis; in particular, Spy, which is essential for the postnatal mitotic proliferation of spermatogonia. This deletion interval is approximately 1-2 Mb and contains eight known genes. In this paper we report the construction of YAC transgenic mice containing different regions of the delta Sxr(b) interval including Zfy1, Ube1y, Smcy, and Eif2s3. Two male and one female founder mice, transgenic for all four genes, were sterile. However, a fertile transgenic, carrying a full-length copy of the Smcy gene integrated into central Chr 12, was identified. Smcy is a highly conserved Y chromosome-located gene, encoding peptides corresponding to epitopes of the male-specific antigen, H-Y. The Smcy transgene was ubiquitously expressed in all organs and tissues tested in male and female carriers. Introduction of the transgene into an X Sxr(b)/O genetic background did not rescue the early arrest of spermatogenesis characteristic of these males. These data indicate that the presence of Smcy is not sufficient to restore spermatogenesis, making it a highly unlikely candidate for Spy.

STSs designed to the retina/pineal-expressed EST clusters THC220430 and THC90422 were originally mapped to 17p13.3 (ref. 7) near a retinitis pigmentosa (RP13) candidate region8. Further testing refined the localization to 17p13.1, between SHGC-2251 and SHGC-6095, within the LCA4 candidate region and approximately 2.5 Mb distal to GUCY2D. Fluorescence in situ hybridization (Fig. 1) confirmed the localization. Fig. 1 Fluorescence in situ hybridization (FISH). AIPL1-containing bacterial artificial chromosome (BAC), shown in red, hybridizes to 17p13.1, consistent with placement of AIPL1 in the Stanford G3 radiation hybrid panel. These data refute the original placement ... cDNA sequencing of the two clusters indicated that the ESTs represent transcripts of one gene. THC90422 transcripts bypass the THC220430 polyadenylation signal, resulting in a 3′ UTR longer by 709 bp. The 180-bp 5′ UTR and coding sequence encoded by the six-exon gene are identical in the 1,538-bp and 2,247-bp transcripts (Fig. 2a). Fig. 2 Gene and protein structure of AIPL1. a, AIPL1 consists of six exons, with alternate polyadenylation sites in the 3′ UTR shown by arrows. Cys239Arg denotes the location of the TGC→CGC missense mutation in exon 5 of the RFS128 family. Trp278X ... The protein encoded by AIPL1 was named human aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) due to its similarity (49% identity, 69% positive) to human aryl hydrocarbon receptor-interacting protein (AIP), a member of the FK506-binding protein (FKBP) family9 (Fig. 2b). The predicted protein consists of 384 amino acids, with a 43,865-dalton molecular mass, and a 5.57 pI. The protein sequence includes three tetratricopeptide repeats (TPR), a 34–amino-acid motif found in proteins with nuclear transport or protein chaperone activity9. Northern-blot hybridization identified mRNA molecules of the predicted sizes in total retinal RNA. The probe also cross-hybridized to 18s rRNA (Fig. 3) in the retina. We detected a weaker signal in skeletal muscle and heart on a poly(A)+ RNA multi-tissue northern blot after very long exposure. It is likely that this signal represents cross-hybridization, as the transcripts differ in size from the retinal mRNAs and are faint. The northern-blot did not indicate AIPL1 expression in brain, but only cerebral tissue was included in the blot. In situ hybridization indicates expression in rat and mouse pineal gland, a high level of expression in adult mouse photoreceptors (Fig. 4) and no expression in cornea (data not shown). Fig. 3 Expression of AIPL1 in human tissues. Northern blots from adult tissues were incubated with an AIPL1 probe. Total retinal RNA blot (top left) and poly(A)+ RNA multi-tissue northern (MTN, top right) are shown. No signal was observed in MTN at 4-, 24- or ... Fig. 4 Retina and pineal expression of Aipl1. a, Digoxygenin in situ hybridization of Aipl1 in adult mouse retina, with expression throughout the outer nuclear layer and photoreceptor inner segments. b, Sense control of (a) with same reaction time. A slight ... Sequencing of the rat Aipl1 cDNA revealed amino acid sequence conservation (87% identity and 96% similarity) between rat and human AIPL1. Rat Aipl1, mouse Aip and human AIP lack a 56–amino-acid carboxy-terminal extension present in AIPL1 (Fig. 2b). This extension includes a ‘hinge’ motif of high flexibility, with multiple O-glycosylation sites, and a casein kinase II (CK2) phosphorylation site, which may be involved in protein complex regulation (as is the CK2 site within the hinge of another FKBP family member, FKBP52; ref. 10). The hinge appears to be conserved in primates, as it is also present in the squirrel monkey (Saimiri sciureus; data not shown). Single-stranded conformational analysis (SSCA) identified three benign nucleotide substitutions within the AIPL1 exon 3 amplimer: G/A at −14, G/A at −10 bp and G/A at codon 100 (Leu100Leu, CTG/CTA). We identified four haplotypes for the combined polymorphisms; the most common, GCG and GAA, have frequencies of 55% and 41%, respectively. Sequencing of AIPL1 from the DNA of one affected individual of the original LCA4 family (Fig. 5a) revealed a homozygous nonsense mutation (Trp278X, TGG→TGA). This allele, if expressed, encodes a protein shorter by 107 amino acids than wild-type AIPL1. The truncated protein includes only 20 of the 34 amino acids of the third TPR motif, a region conserved between human, rat and mouse AIPL1, and AIP. SSCA in other family members confirmed that all affected family members are homozygous for this mutation (Fig. 5a) and that 100 ethnically matched controls did not carry this mutation. Fig. 5 Pedigrees and mutation screen of AIPL1 in families. a, The Trp278X mutation is homozygous in three families: KC, MD and RFS127. SSCA of all living individuals of the KC pedigree demonstrate segregation of the mutant allele. Top electropherogram, an unaffected ... AIPL1 was next analysed in another Pakistani family, MD (Fig. 5a), whose LCA had been mapped to 17p13.1, with GUCY2D excluded by mutational analysis. Sequencing of AIPL1 indicated that affected individuals of this family are homozygous for the Trp278X mutation (Fig. 5a). The MD and KC families differ in haplotype (GCG and GAA, respectively) of the AIPL1 exon 3 polymorphisms, as well as for microsatellite markers tightly linked to AIPL1. These results suggest that the Trp278X mutations causing the LCA in these two families are not derived from a recent, common ancestor. Assay of AIPL1 in 14 families of European descent with LCA that had not been tested previously for linkage to 17p identified apparent disease-causing mutations in three additional families, as follows. Direct sequencing of AIPL1 in the two affected individuals of family RFS121 indicated two mutations, a 2-bp deletion in codon 336 (Ala336Δ2 bp; Fig. 5b) and Trp278X. The deletion results in a frameshift and a termination delayed by 47 codons. The termination signal used in the deletion transcript is upstream of the first AIPL1 polyadenylation signal; therefore, the alternate transcripts from this allele are not predicted to encode alternate proteins. Allele-specific PCR in one affected individual confirmed that the 2-bp deletion and Trp278X are on opposite chromosomes. Therefore, the affected individuals in RFS121 are compound heterozygotes, having received the Trp278X mutation from one parent and the Ala336Δ2 mutation from the other. No unaffected RFS121 family members inherited both mutations. The Ala336Δ2 bp mutation was not observed in 55 unrelated control individuals of European descent. AIPL1 sequencing in two affected individuals from family RFS127 (Fig. 5a) indicated homozygous Trp278X mutations—the same mutation identified in the KC and MD families. Haplo-type analysis of tightly linked microsatellite markers and of the AIPL1 exon 3 polymorphisms suggest that the mutations in the RFS127 and MD families are likely to have descended from a common ancestor; however, there is no indication of Pakistani origin for members of this family. The three affected individuals of family RFS128 (Fig. 5c) are homozygous for a T→C nucleotide substitution predicted to encode a Cys239Arg substitution. This cysteine is conserved in human and rat AIPL1, and in AIP (Fig. 2). This mutation was not identified in over 55 ethnically matched control individuals. Affected members of this family are homozygous for microsatellite markers D17S796 and D17S1881, which are tightly linked, flanking markers of AIPL1. In contrast, affected family members are heterozygous for microsatellite markers D17S960 and D17S1353, which flank GUCY2D. We have identified a new gene that causes LCA4. We detected homozygous AIPL1 mutations in three families in which GUCY2D was excluded as the cause of the disease by linkage or mutation screening: KC, MD and RFS128. AIPL1 is the fourth gene to be associated with LCA. Mutations in AIPL1 may be a common cause of LCA, as an AIPL1 mutation was identified as the apparent cause of the retinal disease in 3 of 14 (21±8%, 90% C.I.) unmapped LCA families. AIPL1 should be assayed in LCA families whose disease locus maps to 17p13 but do not carry disease-causing mutations in GUCY2D, as in 7 of 15 original LCA1 families4. Due to the proximity of AIPL1 and GUCY2D on 17p13, linkage mapping may not distinguish between the genes. Further, is possible that LCA patients who are identical by descent (IBD) at one locus are also IBD at the other. Therefore, both AIPL1 and GUCY2D should be screened for mutations in families whose LCA locus maps to 17p13 or in families with affected individuals who are homozygous for mutations in either gene, unless linkage excludes one of the genes. Of the five families reported here, GUCY2D was excluded by linkage testing or mutation screening in three, the fourth is a compound heterozygote and the fifth is homozygous for a disease-causing mutation confirmed in other families. The similarity of AIPL1 to AIP and the presence of three TPR motifs suggest that it may be involved in retinal protein folding or trafficking. Its role in the pineal gland is also uncertain. The pineal gland contributes to resetting circadian rhythm by diurnal release of melatonin. Additionally, children with destructive pinealomas often display precocious puberty, suggesting a role in long-term periodicity11. Because LCA patients with AIPL1 mutations have grossly abnormal photoreceptors at an early age, the pineal gland also may be affected. Careful clinical characterization of LCA4 patients may reveal pineal-associated abnormalities. Therefore, identifying the exact role of AIPL1 in photoreceptors and the pineal gland will improve our understanding of disease pathology in these patients, and contribute to our understanding of the biology of normal vision and pineal activity.

Visceral left-right asymmetry occurs in all vertebrates, but the inversion of embryo turning (inv) mouse, which resulted following a random transgene insertion, is the only model in which these asymmetries are consistently reversed. We report positional cloning of the gene underlying this recessive phenotype. Although transgene insertion was accompanied by neighbouring deletion and duplication events, our YAC phenotype rescue studies indicate that the mutant phenotype results from the deletion. After extensively characterizing the 47-kb deleted region and flanking sequences from the wild-type mouse genome, we found evidence for only one gene sequence in the deleted region. We determined the full-length 5.5-kb cDNA sequence and identified 16 exons, of which exons 3-11 were eliminated by the deletion, causing a frameshift. The novel gene specifies a 1062-aa product with tandem ankyrin-like repeat sequences. Characterization of complementing and non-complementing YAC transgenic families revealed that correction of the inv mutant phenotype was concordant with integration and intact expression of this novel gene, which we have named inversin (Invs).

Carnitine palmitoyltransferase I (CPT-I) is a key enzyme involved in the regulation of fatty acid oxidation. CPT-IA and CPT-IB are isoforms of carnitine palmitoyltransferase I, of which CPT-IA is expressed in liver, kidney, fibroblasts, and heart and CPT-IB is expressed in skeletal muscle, heart, brown and white adipocytes, and testes. Although the genomic DNA sequence of human CPT-IB is available, the transcription start site and upstream regulatory sequences are not known. For rat CPT-IB, only the cDNA sequence has been published. We have cloned the entire rat CPT-IB gene from a Lambda Fix II rat kidney genomic library. The genomic structure contains 19 exons, with the transcription start site for CPT-IB located in a short first exon, which is a 13-bp extension to the previously published cDNA 5′ sequence. The coding sequence is identical with the rat muscle cDNA. The rat CPT-IB gene contains 18 introns and 19 exons, the latter 18 exons showing 85% homology to the human CPT-IB cDNA. CPT-IB maps to rat chromosome 7 at band q34. A putative promoter region was identified to within 391 bp of the transcription start site. The muscle specificity of the 5′ flanking region was verified by comparison of luciferase expression to that of β-galactosidase in cardiac myocytes and in HepG2 cells.

Fluorescent in situ hybridization (FISH) using a pool of DNA clones that have been mapped to the short arm of chromosome 18 is described. This arm-specific painting technique was used to analyze the chromosomes of two patients who displayed different clinical pictures. One patient had clinical features consistent with tetrasomy 18p, and the other patient had some of the clinical features associated with tri-somy 18. The initial karyotype of both patients showed the presence of a small metacentric marker chromosome, thought to resemble an iso(18p). This interpretation of the karyotype was confirmed for both cases using a specific chromosome arm-painting strategy. While whole-chromosome paints may identify the origin of markers or detect translocations, arm-specific libraries will permit detailed analysis of chromosomal rearrangements when more precise information may be needed.