An essential receptor for adeno-associated virus infection.

Adeno-associated virus (AAV) vectors are currently the leading candidates for virus-based gene therapies because of their broad tissue tropism, non-pathogenic nature and low immunogenicity. They have been successfully used in clinical trials to treat hereditary diseases such as haemophilia B (ref. 2), and have been approved for treatment of lipoprotein lipase deficiency in Europe. Considerable efforts have been made to engineer AAV variants with novel and biomedically valuable cell tropisms to allow efficacious systemic administration, yet basic aspects of AAV cellular entry are still poorly understood. In particular, the protein receptor(s) required for AAV entry after cell attachment remains unknown. Here we use an unbiased genetic screen to identify proteins essential for AAV serotype 2 (AAV2) infection in a haploid human cell line. The most significantly enriched gene of the screen encodes a previously uncharacterized type I transmembrane protein, KIAA0319L (denoted hereafter as AAV receptor (AAVR)). We characterize AAVR as a protein capable of rapid endocytosis from the plasma membrane and trafficking to the trans-Golgi network. We show that AAVR directly binds to AAV2 particles, and that anti-AAVR antibodies efficiently block AAV2 infection. Moreover, genetic ablation of AAVR renders a wide range of mammalian cell types highly resistant to AAV2 infection. Notably, AAVR serves as a critical host factor for all tested AAV serotypes. The importance of AAVR for in vivo gene delivery is further highlighted by the robust resistance of Aavr(-/-) (also known as Au040320(-/-) and Kiaa0319l(-/-)) mice to AAV infection. Collectively, our data indicate that AAVR is a universal receptor involved in AAV infection.

Sex reversal caused by Mus musculus domesticus Y chromosomes linked to variant expression of the testis-determining gene Sry.

When the Y chromosomes from certain populations of Mus musculus domesticus are introduced into the mouse strain C57BL/6 (B6), testis determination can fail, resulting in gonads developing either as ovotestes (with both ovarian and testicular components) or as ovaries. Not all Y(DOM) chromosomes cause sex reversal. Y(DOM) chromosomes are divided into three classes based upon their ability to induce testes in B6. The molecular basis underlying the three Y(DOM) classes is an enigma. The simplest explanation is that they harbor different alleles of the testis-determining gene, Sry. Sequencing of Sry(DOM) genes has indeed identified polymorphisms. However, none were unequivocally linked to the sex-reversal trait. It was concluded that all SRY(DOM) proteins are functionally equivalent. Using a semiquantitative RT-PCR assay, we now show that representatives of the three Y(DOM) classes have variant Sry expression patterns, that severity of sex reversal correlates with Sry mRNA titers, and that genetic correction of the sex reversal results in the upregulation of Sry expression. We propose that the variant Sry expression patterns result from polymorphisms at the site of a putative Sry enhancer.

Ovotestes in B6-XXSxr sex-reversed mice.

The sex-reversed mutation Sxr results in XX males. In the absence of any other mutations, testis differentiation in XXSxr fetuses is essentially normal and only one report of an XXSxr fetus with ovotestes is in the literature. We report that 84% (21/25) of 13 days postcoitum XXSxr fetuses on the B6 inbred genomic background have ovotestes. Ovotestes were found in fetuses from both Sxra and Sxrb variants. Examination of fetuses older than 13 dpc suggests that the presence of ovotestes is transient in most fetuses. However, one overt hermaphrodite was identified after birth. The development of ovotestes is associated with the inbred background and is exacerbated by the dominant spotting oncogene allele KitW-42J. We propose that spreading of X-inactivation into the Sxr region resulting in loss of Sry expression is more extensive in B6-Sxr strains.

The dominant white spotting oncogene allele Kit(W-42J) exacerbates XY(DOM) sex reversal.

The Y chromosome from certain populations of M. m. domesticus is incapable of normal testis determination in the B6 inbred strain resulting in XY hermaphrodites or XY females (XY(DOM) sex reversal). B6 consomic strains have been developed with either transient (B6-Y(AKR)) or severe (B6-Y(TIR)) XY(DOM) sex reversal. We report that a point mutation, the dominant white spotting oncogene allele, Kit(W-42J), exacerbates XY(DOM) sex reversal. In B6-Y(AKR), penetrance of the trait is low; however, in B6-Y(TIR), Kit(W-42J) exacerbated sex reversal to such an extent that almost all XY progeny developed into females. The exacerbation of sex reversal was not linked to retardation of early fetal growth or reduction of testis size. Furthermore, semiquantitative RT-PCR for the testis-determining gene, Sry, suggests that exacerbation of sex reversal in B6-Y(TIR) is not due to blockade of Sry expression, a substantial delay in initiation of Sry expression, or exceptionally low levels of Sry mRNAs. We propose that Kit(W-42J) enhances sex reversal by adversely affecting a critical step in testis differentiation that is downstream of Sry.

Absence of correlation between Sry polymorphisms and XY sex reversal caused by the M. m. domesticus Y chromosome.

Mus musculus domesticus Y chromosomes (YDOM Chrs) vary in their ability to induce testes in the strain C57BL/6J. In severe cases, XY females develop (XYDOM sex reversal). To identify the molecular basis for the sex reversal, a 2.7-kb region of Sry, the testis-determining gene, was sequenced from YDOM Chrs linked to normal testis determination, transient sex reversal, and severe sex reversal. Four mutations were identified. However, no correlation exists between these mutations and severity of XYDOM sex reversal. RT-PCR identified Sry transcripts in XYDOM sex-reversed fetal gonads at 11 d.p.c., the age when Sry is hypothesized to function. In addition, no correlation exists between XYDOM sex reversal and copy numbers of pSx1, a Y-repetitive sequence whose deletion is linked to XY sex reversal. We conclude that SRY protein variants, blockade of Sry transcription, and deletion of pSx1 sequences are not the underlying causes of XYDOM sex reversal.

The testis-determining gene, SRY, exists in multiple copies in Old World rodents.

SRY is a unique gene on the Y chromosome in most mammalian species including the laboratory mouse, Mus musculus, and the closely related European wild mouse species M. spicilegus, M. macedonicus, and M. spretus. In contrast, SRY is present in 2-6 copies in the more distantly related Asian mouse species M. caroli, M. cervicolor, and M. cookii and in 2-13 copies in the related murid species Pyromys saxicola, Coelomys pahari, Nannomys minutoides, Mastomys natalensis, and Rattus norvegicus. Copy numbers do not correlate with known phylogenetic relationships suggesting that SRY has undergone a rapid and complex evolution in these species. SRY was recently proposed as a molecular probe for phylogenetic inferences. The presence of multiple SRY genes in a wide range of murid species and genera, and at least one cricetid species, necessitates caution in the use of SRY for phylogenetic studies in the Rodentia unless it is ascertained that multiple SRY genes do not exist.

Distribution of the molossinus allele of Sry, the testis-determining gene, in wild mice.

When the Y chromosome of the laboratory inbred mouse strain C57BL/6 (B6) is replaced by the Y of certain strains of Mus musculus domesticus, testis determination fails and all XY fetuses develop either as hermaphrodites or XY females (XY sex reversal). This suggests the presence of at least two alleles of Sry, the male-determining gene on the Y:M. m. domesticus and B6. The B6 Y chromosome is derived from the Japanese house mouse, M. m. molossinus and therefore carries a molossinus Sry allele. As a first step to determine how the molossinus Sry allele evolved, its distribution pattern was determined in wild mice. The cumulative data of 96 M. musculus samples obtained from 58 geographical locations in Europe, North Africa, and Asia show the molossinus Sry allele is restricted to Japan and the neighboring Asian mainland and confirm that Japanese M. m. molossinus mice were derived in part from a race of M. m. musculus from Korea or Manchuria. Sry polymorphisms, as illustrated by the molossinus Sry allele, can serve as molecular markers for studies on the evolution of wild M. musculus populations and can help determine the role sex determination plays in speciation.

Polymorphism of a CAG trinucleotide repeat within Sry correlates with B6.YDom sex reversal.

Breeding the Y chromosome from certain Mus musculus domesticus strains onto the inbred laboratory mouse strain, C57BL/6J (B6), results in hermaphroditic progeny. This strain-dependent sex reversal suggests that there may be significant allelic variation in the murine sex determining gene, Sry. We have analysed the Sry genes from several domesticus-type Y chromosomes and show that they encode smaller proteins than the molossinus-type alleles SryB6 and Sry129. We have also identified a polymorphic stretch of trinucleotide repeats that is unique to strains causing sex reversal and show that specific changes in the predicted polyglutamine amino acid sequence at this site are associated with different degrees of sex reversal.

The musculus-type Y chromosome of the laboratory mouse is of Asian origin.

Mus musculus domesticus, M.m. bactrianus, M.m. musculus, M.m. castaneus, and M.m. molossinus wild mice were investigated for polymorphisms of the Y Chromosome (Chr) genes Zinc finger-Y (Zfy) and Sex-determining region-Y (Sry). Zfy divided the Y Chrs of these mice into domesticus- (domesticus) and musculus-types (musculus, castaneus, molossinus). M.m. bactrianus specimens had both Y Chrs, possibly owing to the introgression of a musculus-type Y into this population. Sry identified a subpopulation of musculus-type Y chromosomes. This subpopulation, designated the molossinus-type, was found in M.m. molossinus, a M. musculus subspecies specimen from northern China (Changchun), and laboratory mice. The cumulative data suggest that M.m. musculus of northern China and Korea are a subpopulation distinct from M.m. musculus of Europe and central China and that this subpopulation invaded Japan, giving rise to M.m. molossinus. Furthermore, the data suggest that the musculus-type Y of the laboratory mouse originated from this subpopulation, corroborating early historical records reporting that Chinese and Japanese mice that were imported into Europe for the pet trade contributed to the genome of the laboratory mouse.

The two candidate testis-determining Y genes (Zfy-1 and Zfy-2) are differentially expressed in fetal and adult mouse tissues.

The candidate testis-determining Y genes of the mouse Zfy-1 and Zfy-2, encode proteins containing an acidic amino terminus and a carboxyl terminus composed of 13 zinc fingers. The zinc finger domain is conserved among human and mouse zinc finger X and Y genes. We report a 6-amino-acid deletion in the Zfy-2 zinc finger domain of laboratory mice possessing musculus Y chromosomes. The effect of this deletion on the function of Zfy-2 is not known. The reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot techniques were used to study expression of Zfy in adults and fetuses. In adults, the data suggest that Zfy-1 and Zfy-2 transcription is linked to spermatogenesis, that transcription increases with the initiation of meiosis, and that high levels of these mRNAs are found in postmeiotic round spermatid cells. The data also suggest that differential expression of these two genes is present with expression of Zfy-2 being slightly greater than Zfy-1. In fetuses, Zfy transcripts were detected in several tissues, including the testes. In contrast to the situation in adults, the data suggest that expression of Zfy-1 is greater than that of Zfy-2. The data suggesting that Zfy-1 expression is present in fetal testes support the hypothesis that this gene plays a role in testis differentiation. However, because the Zfy genes are apparently also expressed during spermatogenesis and in fetal organs other than testes, they may serve additional functions besides their postulated role in testis determination.

Development and fertility of ovaries in the B6.YDOM sex-reversed female mouse.

When the Y chromosome of Mus musculus domesticus (YDOM) was introduced onto the C57BL/6 (B6) mouse background, half of the XY progeny (B6.YDOM) developed bilateral ovaries and female internal and external genitalia. We examined the fertility of the B6.YDOM sex-reversed female mouse. The chromosomal sex of the individual mouse was identified by dot hybridization with mouse Y chromosome-specific DNA probes. The results indicated that all XY females lacked regular estrous cyclicity although most were able to mate and ovulate after treatment with gonadotropins. When they had been ovariectomized and grafted with ovaries from the XX female litter mate, they initiated estrous cyclicity. Reciprocally, the XX female that had received XY ovarian grafts did not resume estrous cyclicity. Development of the XY ovary was morphologically comparable to the XX ovary until 16 day of gestation (d.g.), when most germ cells had reached the zygotene or pachytene stage of meiotic prophase. However, by the day of delivery (19 or 20 d.g.), no oocyte remained in the medullary cords of the XY ovary. In the control XX ovary, the first generation of follicles developed in the medullary region, and 5 delta-3 beta-hydroxysteroid dehydrogenase (3 beta-HSDH) activity appeared first in the stromal cells around growing follicles by 10 days after birth. In contrast, in the XY ovary, follicles were not formed in the medullary region, and 3 beta-HSDH activity appeared in epithelial cells of the oocyte-free medullary cords. Primordial follicles in the cortex region continued development in both the XX and XY ovaries. These results suggest that the XY female is infertile due to a defect inside the XY ovary. The prenatal loss of oocytes in the medullary cords may be a key event leading to abnormal endocrine function, and thereby, the absence of estrous cyclicity.

Chromosome mapping and expression of a putative testis-determining gene in mouse.

Isolation and mapping of a mouse complementary DNA sequence (mouse Y-finger) encoding a multiple, potential zinc-binding, finger protein homologous to the candidate human testis-determining factor gene is reported. Four similar sequences were identified in Hind III-digested mouse genomic DNA. Two (7.2 and 2.0 kb) were mapped to the Y chromosome. Only the 2.0-kb fragment, however, was correlated with testis determination. Polymerase chain reaction analysis suggests both Y loci are transcribed in adult testes. A 3.6-kb fragment was mapped to the X chromosome between the T16H and T6R1 translocation breakpoints, and a fourth (6.0 kb) was mapped to chromosome 10. Hence, mYfin sequences have been duplicated several times in the mouse, although they are not duplicated in humans.

Linkage of the murine steroid sulfatase locus, Sts, to sex reversed, Sxr: a genetic and molecular analysis.

We present genetic and molecular data demonstrating linkage of the gene for steroid sulfatase (Sts) to the mutation sex reversed (Sxr) definitively showing the existance of a functional allele for Sts mapping to the pseudoautosomal region of the mouse Y chromosome. Thus, in mouse, functional Sts genes are present in the pseudoautosomal region of both the X and Y chromosomes. This is in contrast to man where Sts has been mapped to the short arm of the X just centromeric to the pseudoautosomal region. Only a single recombinant separating Sts and Sxr was found out of 103 male meioses analyzed; double recombinants were not found between sex (Tdy), Sts and Sxr. If the rate of recombination in the pseudoautosomal region in male mice is equivalent to that in man and thus 7-10X higher than normal, then our data suggest that the distance between Sts and Sxr (or the telomere of the Y) is approximately 100-200 kb in length. Our data is in contrast to a recent report of a recombination frequency separating Sts and Sxr of as high as 6.2-9.8%.

Morphological development of the mouse gonad in tda-1 XY sex reversal.

tda-1 XY sex reversal occurs when the Y chromosome of at least some populations of wild Mus musculus domesticus is placed on the C57BL/6J genomic background. Gross anatomical observations have previously revealed morphological similarities among fetal ovotestes of tda-1 and Tas-inherited XY sex reversals and BALB/cWt mosaic hermaphrodites. We studied the histology of tda-1 XY sex-reversed gonads, ranging in age from day 14 of gestation to adult. The obtained data revealed additional similarities with ovotestes of BALB/cWt mosaic hermaphrodites as well as with ovotestes of hermaphrodites found in XXSxr and XX/XY chimeras. It is proposed that ovotestes occurring in these various hermaphroditic conditions may be formed through a common pathway.

Studies on the genetics of tda-1 XY sex reversal in the mouse.

When the Y chromosome of at least some populations of the house mouse of Western Europe and the Mediterranean, Mus musculus domesticus, is placed into the C57BL/6J (B6) inbred mouse genome, XY fetuses develop into hermaphrodites or females. It has been hypothesized that the testis-determining gene on the Y chromosome of M. m. domesticus (TdyDOM) interacts improperly with a putative B6/J recessive, testis-determining, autosomal gene (tda-1). The present study extended these earlier findings. The mating of B6 mice possessing the Y chromosome of M. m. domesticus (B6.YDom/Na; N6-N9) to females of the AKR, BALB/c, C3H/An, and C3H/He, but not SJL, strains resulted in aberrant testicular differentiation in day-14/15 F1 fetuses. The aberrant testes were characterized by a delay in testicular differentiation at the cranial and caudal poles of the gonad, i.e., the presence of a thin (or no) tunica albuginea and the presence of disorganized (or no) seminiferous tubules. Crossing B6.YDom male phenotypes with SJL females did not result in aberrant testicular differentiation, suggesting that the SJL strain possesses the dominant testis-determining, autosomal-1 allele, Tda-1. Studies using recombinant DNA probes specific for the murine Y chromosome have suggested that the SJL and AKR strains possess the M. m. domesticus Y chromosome. When Y chromosomes of the SJL and AKR strains were placed on the B6 background, aberrant testicular differentiation similar to tda-1 XY sex reversal occurred in only 1 out of 87 (1%) N4 day-14/15 fetuses possessing YSJL, but in 25 out of 45 (56%) N4 day-14/15 fetuses possessing YAKR.(ABSTRACT TRUNCATED AT 250 WORDS)

H-Y antigen in XO mice.

We examined the expression of H-Y antigen in 14 XO female mice using three monoclonal H-Y-specific antibodies. We found that spleen and liver cells from XO mice removed the reactivity of these antibodies at the same efficacy as XY cells. However radiobinding assays on cultured XO cells suggested a qualitative or quantitative difference between XO and XY cells. In cell-mediated cytolysis (CMC), H-Y-specific reactivity was observed when XO fibroblasts were used as targets, but no reactivity was observed when XO concanavalin A (Con A) blasts were used as targets. We concluded from these studies that XO mice do express H-Y antigen, detected both by serologic assays and cell-mediated assays.