Alterations in chromatin at antigen receptor loci define lineage progression during B lymphopoiesis.

Developing lymphocytes diversify their antigen receptor (AgR) loci by variable (diversity) joining (V[D]J) recombination. Here, using the micrococcal nuclease (MNase)-based chromatin accessibility (MACC) assay with low-cell count input, we profile both small-scale (kilobase) and large-scale (megabase) changes in chromatin accessibility and nucleosome occupancy in primary cells during lymphoid development, tracking the changes as different AgR loci become primed for recombination. The three distinct chromatin structures identified in this work define unique features of immunoglobulin H (IgH), Igκ, and T cell receptor-α (TCRα) loci during B lymphopoiesis. In particular, we find locus-specific temporal changes in accessibility both across megabase-long AgR loci and locally at the recombination signal sequences (RSSs). These changes seem to be regulated independently and can occur prior to lineage commitment. Large-scale changes in chromatin accessibility occur without significant change in nucleosome density and represent key features of AgR loci not previously described. We further identify local dynamic repositioning of individual RSS-associated nucleosomes at IgH and Igκ loci while they become primed for recombination during B cell commitment. These changes in chromatin at AgR loci are regulated in a locus-, lineage-, and stage-specific manner during B lymphopoiesis, serving either to facilitate or to impose a barrier to V(D)J recombination. We suggest that local and global changes in chromatin openness in concert with nucleosome occupancy and placement of histone modifications facilitate the temporal order of AgR recombination. Our data have implications for the organizing principles that govern assembly of these large loci as well as for mechanisms that might contribute to aberrant V(D)J recombination and the development of lymphoid tumors.

The murine IgH locus contains a distinct DNA sequence motif for the chromatin regulatory factor CTCF.

Antigen receptor assembly in lymphocytes involves stringently-regulated coordination of specific DNA rearrangement events across several large chromosomal domains. Previous studies indicate that transcription factors such as paired box 5 (PAX5), Yin Yang 1 (YY1), and CCCTC-binding factor (CTCF) play a role in regulating the accessibility of the antigen receptor loci to the V(D)J recombinase, which is required for these rearrangements. To gain clues about the role of CTCF binding at the murine immunoglobulin heavy chain (IgH) locus, we utilized a computational approach that identified 144 putative CTCF-binding sites within this locus. We found that these CTCF sites share a consensus motif distinct from other CTCF sites in the mouse genome. Additionally, we could divide these CTCF sites into three categories: intergenic sites remote from any coding element, upstream sites present within 8 kb of the VH-leader exon, and recombination signal sequence (RSS)-associated sites characteristically located at a fixed distance (∼18 bp) downstream of the RSS. We noted that the intergenic and upstream sites are located in the distal portion of the VH locus, whereas the RSS-associated sites are located in the DH-proximal region. Computational analysis indicated that the prevalence of CTCF-binding sites at the IgH locus is evolutionarily conserved. In all species analyzed, these sites exhibit a striking strand-orientation bias, with >98% of the murine sites being present in one orientation with respect to VH gene transcription. Electrophoretic mobility shift and enhancer-blocking assays and ChIP-chip analysis confirmed CTCF binding to these sites both in vitro and in vivo.

Altered expression of microRNA miR-146a correlates with the development of chronic renal inflammation.

MicroRNAs (miRNAs) are highly conserved small non-coding RNAs that act as post-transcriptional regulators of target mRNA. In this study, we sought to identify the microRNA underlying local inflammation in a murine model of chronic kidney disease (CKD). In microarray analysis of kidneys, the expression of miR-146a/b was elevated in B6.MRLc1 CKD mice that spontaneously develop renal inflammation with age. Primary-microRNA analysis found that elevated miR-146a/b expression in the kidneys of B6.MRLc1 mice was mainly derived from miR-146a rather than miR-146b, and this expression increased with the development of CKD. Histopathological scores for glomerular and interstitial lesions, mRNA expression of inflammatory mediators, and macrophage infiltration were significantly higher in B6.MRLc1 than C57BL/6 mice and were positively correlated with miR-146a expression. In situ hybridization and laser microdissection-RT-PCR showed that miR-146a expression in interstitial lesions containing inflammatory cells was higher than in the glomerulus. The increased expression of the inflammatory-associated genes RELA, IRAK1, IL1B, IL10, and CXCLs was noted in miR-146a/b-silenced human monocytes. The amount of miR-146a was higher in urine sediments of B6.MRLc1 than of C57BL/6 mice. Thus, miR-146a expression in the kidneys and its urinary excretion was specifically associated with the development of interstitial lesions and correlated with inflammatory cell infiltration.

Genomic analysis of the appearance of testicular oocytes in MRL/MpJ mice.

Mammals produce sperm or oocytes depending on their sex; however, newborn MRL/MpJ (MRL) male mice produce oocytes within their testes. We previously reported that one of the genes responsible for this phenotype is present on the MRL-type Y chromosome (Y(MRL)), and that multiple genes, probably autosomal, are also required for the development of this phenotype. In this study we focused on the autosomal genes and examined their relationship with this phenotype by analyzing the progeny from crosses between MRL mice and other strains. We first observed the male F1 progeny from the crosses between female A/J, C57BL/6 (B6), BALB/c, C3H/He, or DBA/2 mice and male MRL mice, and two consomic strains, male B6-Y(MRL) and MRL-Y(B6). Testicular oocytes that were morphologically similar to those of MRL mice were detected in all mouse strains except BALBMRLF1; however, the incidence of testicular oocytes was significantly lower than that in MRL mice. The appearance of testicular oocytes in MRL-Y(B6) mice indicates that this phenotype is strongly affected by genomic factors present on autosomes, and that there is at least one other causative gene on the MRL-type autosomes (MRL testicular oocyte production, mtop) other than that on Y(MRL). Furthermore, a quantitative trait locus (QTL) analysis using N2 backcross progeny from crosses between female MRLB6F1 and male MRL mice revealed the presence of susceptibility loci for the appearance of testicular oocytes at 8-17 cM on Chr 15. These findings demonstrate that the appearance of testicular oocytes is regulated by the genetic factors on Chr 15 and on Y(MRL).

Ovarian cysts in MRL / MpJ mice are derived from the extraovarian rete: a developmental study.

MRL/MpJ (MRL) mice, commonly used as a model for autoimmune disease, have a high frequency of ovarian cysts originating from the rete ovarii. In the present study, to clarify how the rete ovarii, which are remnants of mesonephric tubules during embryogenesis, progress to cystic formation with aging, the morphology of MRL rete ovarii was analyzed and compared with that of normal C57BL/6N (B6) mice. In B6 mice, the rete ovarii consisted of a series of tubules, including the extraovarian rete (ER), the connecting rete (CR), and the intraovarian rete (IR), based on their location. Whereas the ER of B6 mice was composed of highly convoluted tubules lined by both ciliated and non-ciliated epithelia, the tubules in the CR and IR had only non-ciliated cells. In MRL mice, dilations of the rete ovarii initiated from the IR rather than the ER or CR. Although the histological types of cells lining the lumen of the rete ovarii were the same as those in B6 mice, the ER in MRL mice showed a variety in morphology. In particular, the connections between the ER and ovary tended to disappear with increasing age and the development of ovarian cysts. Furthermore, the epithelium lining the large ovarian cysts in MRL mice had ciliated cells forming the cluster. On the basis of these findings, it is suggested that cystic changes of the rete ovarii in MRL mice are caused by the dilations of the IR with invasion of the ER and CR into the ovarian medulla. These data provide new pathological mechanisms for ovarian cyst formation.

Hepatocyte nuclear factor 4 alpha is associated with mesenchymal-epithelial transition in developing kidneys of C57BL/6 mice.

During kidney development, the metanephric mesenchyme (MM) develops into the nephron through mesenchymal-epithelial transition (MET). We have previously reported that knock-down of the expression of hepatocyte nuclear factor 4 alpha (Hnf4a) gene induces failure of cellular organization in the condensed mesenchyme (CM) of cultured embryonic kidneys. To elucidate the details of MET during nephrogenesis, embryonic mouse kidneys were analyzed by electron microscopy, immunohistochemistry, and molecular biology. The findings showed that the intercellular junction, but not the basal lamina, was present in the CM. Additionally, immediately after Hnf4a gene expression, the expression of epithelial genes (Krt8, Tjp1, and Cdh1) increased, and those of mesenchymal genes (Acta1 and Vim) decreased, in the CM compared to the MM. To clarify the relationship between MET and Hnf4α, the fibroblast cell line with forced expression of Hnf4α protein were analyzed. In this model, it was noted that Hnf4α induced increasing epithelial and decreasing mesenchymal gene expression. In these, up-regulation of Pvrl1, -2, and Mllt4 genes which mediate the formation of apico-basal polarity, were found. These results, and those of previous findings, indicate that Hnf4α protein is associated with the initiation of MET in early nephrogenesis.

Identifying a new locus that regulates the development of rete ovarian cysts in MRL/MpJ mice.

MRL/MpJ (MRL) is a mouse model for autoimmune disease and develops ovarian cysts with age. The ovarian cysts originate from the rete ovarii, which is considered to be the remnant of fetal mesonephric tubules. In a previous study, we analyzed the genetic background of ovarian cysts by using backcross progenies between MRL and C57BL/6N (B6) mice. By interval mapping, suggestive linkages were detected on several chromosomes (Chrs), and a significant linkage on Chr 14 was designated as MRL Rete Ovarian Cyst (mroc). In the present study, which evaluated 113 F2 intercross progenies, a significant linkage appeared on Chr 6 at the marker position D6Mit188 (likelihood ratio statistic = 18.5). In particular, the peak regions of Chrs 6 and 14, which contain major causative loci by backcross analysis, showed close reverse interaction. From these results, a locus on Chr 6 was identified as mroc2, the second major locus associated with ovarian cyst formation in MRL mice.

Analysis of factors decreasing testis weight in MRL mice.

MRL/MpJ (MRL) mouse testes have several unique characteristics, including the appearance of oocytes, the occurrence of metaphase-specific apoptosis of meiotic spermatocytes, and the presence of heat-shock-resistant spermatocytes. In the present study we used chromosomal mapping to determine the genomic background associated with small testis size in MRL mice. We prepared and analyzed C57BL/6-based congenic mice carrying MRL mouse loci. Quantitative trait loci (QTL) analysis revealed susceptibility loci for small testis size at 100 cM on chromosome (Chr) 1 and at around 80 cM on Chr 2. Analysis with B6.MRLc1 and B6.MRLc2 congenic mice and double-congenic mice confirmed the QTL data and showed that low testis weight in MRL mice was caused by germ cell apoptosis. Through histological examinations we found that B6.MRLc1 and B6.MRLc2 mice showed stage-specific apoptosis in their testes, the former at metaphase stage XII and the later at pachytene stage IV. Metaphase-specific apoptosis of spermatocytes occurs due to mutation of the exonuclease 1 (Exo1) gene located at 100 cM on Chr 1. Thus, the mutation of the Exo1 gene is also responsible for low testis weight caused by metaphase-specific apoptosis. In conclusion, testis weight is reduced in MRL mice due to apoptosis of germ cells caused by mutations in loci on Chrs 1 and 2.

TOX Regulates Growth, DNA Repair, and Genomic Instability in T-cell Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes. Using a transgenic screen in zebrafish, thymocyte selection-associated high mobility group box protein (TOX) was uncovered as a collaborating oncogenic driver that accelerated T-ALL onset by expanding the initiating pool of transformed clones and elevating genomic instability. TOX is highly expressed in a majority of human T-ALL and is required for proliferation and continued xenograft growth in mice. Using a wide array of functional analyses, we uncovered that TOX binds directly to KU70/80 and suppresses recruitment of this complex to DNA breaks to inhibit nonhomologous end joining (NHEJ) repair. Impaired NHEJ is well known to cause genomic instability, including development of T-cell malignancies in KU70- and KU80-deficient mice. Collectively, our work has uncovered important roles for TOX in regulating NHEJ by elevating genomic instability during leukemia initiation and sustaining leukemic cell proliferation following transformation.Significance: TOX is an HMG box-containing protein that has important roles in T-ALL initiation and maintenance. TOX inhibits the recruitment of KU70/KU80 to DNA breaks, thereby inhibiting NHEJ repair. Thus, TOX is likely a dominant oncogenic driver in a large fraction of human T-ALL and enhances genomic instability. Cancer Discov; 7(11); 1336-53. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1201.

A critical role for PKR complexes with TRBP in Immunometabolic regulation and eIF2α phosphorylation in obesity.

Aberrant stress and inflammatory responses are key factors in the pathogenesis of obesity and metabolic dysfunction, and the double-stranded RNA-dependent kinase (PKR) has been proposed to play an important role in integrating these pathways. Here, we report the formation of a complex between PKR and TAR RNA-binding protein (TRBP) during metabolic and obesity-induced stress, which is critical for the regulation of eukaryotic translation initiation factor 2 alpha (eIF2α) phosphorylation and c-Jun N-terminal kinase (JNK) activation. We show that TRBP phosphorylation is induced in the setting of metabolic stress, leading to PKR activation. Suppression of hepatic TRBP reduced inflammation, JNK activity, and eIF2α phosphorylation and improved systemic insulin resistance and glucose metabolism, while TRBP overexpression exacerbated the impairment in glucose homeostasis in obese mice. These data indicate that the association between PKR and TRBP integrates metabolism with translational control and inflammatory signaling and plays important roles in metabolic homeostasis and disease.

Differences in spermatogenesis in cryptorchid testes among various strains of mice.

The purpose of the study reported here was to define strain differences in spermatogenesis in cryptorchid testes in mice. Mice of strains A/J, BALB/c, CBA/N, C3H/He, C57BL/6 (B6), ddY and ICR were found to be sensitive to heat stress attributable to experimentally induced cryptorchidism. In contrast, mice of strains AKR/N (AKR), MRL/MpJ-+/+ (M+) and MRL/MpJ-lpr/lpr (lpr) were resistant to heat stress. Relative increases of apoptotic cells were detected in the sensitive group, but not in the resistant group. A decrease of proliferating cell nuclear antigen-immunoreactive cells after experimentally induced cryptorchidism was observed only in the sensitive group. These results suggested that heat stress-resistant germ cells were present in MRL and AKR strains, possibly originating from the genetic background.

Expression and distribution of inducible nitric oxide synthase in mouse testis.

Nitric oxide (NO) is a simple and relatively unstable radical under physiological conditions. It is synthesized by three isoforms of NO synthase, that is neuronal, endothelial and inducible (iNOS) isoforms. In the present study, we investigated the distribution of iNOS with immunohistochemical methods in the mouse testis. The iNOS-immunoreactivity was detected on the basal region of the seminiferous tubules, where the cytoplasm of Sertoli cells was selectively immunolabeled. This immunoreactivity was observed by both immunofluorescent and immunoenzyme methods. Weak immunoreactivity was detected on the perinuclear cytoplasm of Sertoli cells throughout the seminiferous stages, whereas in stages I-VIII, it was remarkable on the processes of Sertoli cells surrounding the spermatogonia and early spermatocytes, and elongating into the lumina of seminiferous tubules. By reverse transcriptase-polymerase chain reaction, mRNA for iNOS was found to be expressed in the mouse testis. These results reveal that iNOS is consistently distributed at the front of the testicular environment.

Exon skipping of exonuclease 1 in MRL/MpJ mice is caused by a nucleotide substitution of the branchpoint sequence in intron eight.

In MRL/MpJ mice, there is a genetic mutation of exonuclease 1 (Exo1), in which the exon 9 is sometimes deleted. In the present study, to check the generation of the spliced exons, exon 8-intron 8-exon 9 (pCX/Ex/EIE/B and pCX/Ex/EIE/M) plasmids were temporally transfected in vitro into BALB 3T3 cells, and RT-PCR using appropriate primer pair was carried out 1 day after transfection. In these constructions, pCX/Ex/EIE/B was derived from genomic sequence of C57BL/6 mice, and pCX/Ex/EIE/M was from MRL/MpJ. A spliced band was detected in pCX/Ex/EIE/B, but was present little or very weakly in pCX/Ex/EIE/M. Next, the same spliced band was demonstrated in the pCX/Ex/EIE/M(T) plasmid, in which the branchpoint sequence (BPS) of pCX/Ex/EIE/M including the exon 9 was changed into that of pCX/Ex/EIE/B. The splicing did not occur in the dell1/B mutant, in which 1960 nucleotides of the intron 8 were deleted, whereas it was detected in the del2/B plasmid deleted 1036 nucleotides in its middle region. These results suggest that the nucleotide T to A mutation of the BPS in the intron 8 is at least a sufficient for generation of splice variants (tr-1 and tr-2 Exo1).

Genomic analysis of the appearance of ovarian mast cells in neonatal MRL/MpJ mice.

In MRL/MpJ mice, ovarian mast cells (OMCs) are more abundant than in other mouse strains, and tend to distribute beneath the ovarian surface epithelium at birth. This study investigated the factors regulating the appearance of neonatal OMCs in progeny of the cross between MRL/MpJ and C57BL/6N strains. F1 neonates had less than half the number of OMCs than MRL/MpJ. Interestingly, MRLB6F1 had more neonatal OMCs than B6MRLF1, although they were distributed over comparable areas. Furthermore, in MRL/MpJ fetuses for which parturition was delayed until embryonic day 21.5, the number of OMCs was significantly higher than in age-matched controls at postnatal day 2. These results suggest that the number of OMCs was influenced by the environmental factors during pregnancy. Quantitative trait locus analysis using N2 backcross progeny revealed two significant loci on chromosome 8: D8Mit343-D8Mit312 for the number of OMCs and D8Mit86-D8Mit89 for their distribution, designated as mast cell in the ovary of MRL/MpJ 1 (mcom1) and mcom2, respectively. Among MC migration-associated genes, ovarian expression of chemokine (C-C motif) ligand 17 at mcom1 locus was significantly higher in MRL/MpJ than in C57BL/6N, and positively correlated with the expression of OMC marker genes. These results indicate that the appearance of neonatal OMCs in MRL/MpJ is controlled by environmental factors and filial genetic factors, and that the abundance and distribution of OMCs are regulated by independent filial genetic elements.

Molecular pathology of murine ureteritis causing obstructive uropathy with hydronephrosis.

Primary causes of urinary tract obstruction that induces urine retention and results in hydronephrosis include uroliths, inflammation, and tumors. In this study, we analyzed the molecular pathology of ureteritis causing hydronephrosis in laboratory rodents.F2 progenies of C57BL/6 and DBA/2 mice were studied histopathologically and by comprehensive gene expression analysis of their ureters. Incidence of hydronephrosis was approximately 5% in F2 progenies. Histopathologically, this hydronephrosis was caused by stenosis of the proximal ureter, which showed fibrosis and papillary malformations of the proliferative epithelium with infiltrations of B-cell-dominated lymphocytes. Additionally, CD16-positive large granular leukocytes and eosinophils infiltrated from the ureteral mucosa to the muscular layer. Eosinophilic crystals were characteristically observed in the lumen of the ureter and the cytoplasm of large granular leukocytes, eosinophils, and transitional epithelial cells. Comprehensive gene profiling revealed remarkably elevated expression of genes associated with hyperimmune responses through activation of B cells in diseased ureters. Furthermore, diseased ureters showed dramatically higher gene expression of chitinase 3-like 3, known as Ym1, which is associated with formation both of adenomas in the transitional epithelium and of eosinophilic crystals in inflammatory conditions. The Ym1 protein was mainly localized to the cytoplasm of the transitional epithelium, infiltrated cells, and eosinophilic crystals in diseased ureters.We determined that the primary cause of hydronephrosis in F2 mice was ureteritis mediated by the local hyperimmune response with malformation of the transitional epithelium. Our data provide a novel molecular pathogenesis for elucidating causes of aseptic inflammation in human upper urinary tracts.

Quantitative and qualitative urinary cellular patterns correlate with progression of murine glomerulonephritis.

The kidney is a nonregenerative organ composed of numerous functional nephrons and collecting ducts (CDs). Glomerular and tubulointerstitial damages decrease the number of functional nephrons and cause anatomical and physiological alterations resulting in renal dysfunction. It has recently been reported that nephron constituent cells are dropped into the urine in several pathological conditions associated with renal functional deterioration. We investigated the quantitative and qualitative urinary cellular patterns in a murine glomerulonephritis model and elucidated the correlation between cellular patterns and renal pathology.Urinary cytology and renal histopathology were analyzed in BXSB/MpJ (BXSB; glomerulonephritis model) and C57BL/6 (B6; control) mice. Urinary cytology revealed that the number of urinary cells in BXSB mice changed according to the histometric score of glomerulonephritis and urinary albumin; however, no correlation was detected for the levels of blood urea nitrogen and creatinine. The expression of specific markers for podocytes, distal tubules (DTs), and CDs was detected in BXSB urine. Cells immunopositive for Wilms tumor 1 (podocyte marker) and interleukin-1 family, member 6 (damaged DT and CD marker) in the kidney significantly decreased and increased in BXSB versus B6, respectively. In the PCR array analysis of inflammatory cytokines and chemokines, Il10, Cxcl2, C3, and Il1rn showed relatively higher expression in BXSB kidneys than in B6 kidneys. In particular, the highest expression of C3 mRNA was detected in the urine from BXSB mice. Furthermore, C3 protein and mRNA were localized in the epithelia of damaged nephrons.These findings suggest that epithelial cells of the glomerulus, DT, and CD are dropped into the urine, and that these patterns are associated with renal pathology progression. We conclude that evaluation of urinary cellular patterns plays a key role in the early, noninvasive diagnosis of renal disease.

ATRIP associates with replication protein A-coated ssDNA through multiple interactions.

The ATR (ATM- and rad3-related)-mediated checkpoint pathway has a crucial role in regulating the cellular responses to DNA damage and DNA-replication stress. ATRIP (ATR-interacting protein), the regulatory partner of ATR, binds directly to replication protein A (RPA)-coated ssDNA and enables the ATR-ATRIP complex to recognize this DNA damage-induced structure. Here, we show that ATRIP associates with RPA-ssDNA through multiple interactions. Two major RPA-ssDNA-interacting domains of ATRIP were mapped to the regions flanking the conserved coiled-coil domain. In contrast to a recent article, we found that ATRIP mutants lacking the N terminus retained the ability to bind to RPA-ssDNA, suggesting that the multiple interactions between ATRIP and RPA-ssDNA may function redundantly in the recruitment of ATR-ATRIP. Unexpectedly, one internal region of ATRIP exhibited affinity to ssDNA, suggesting that ATRIP may interact with ssDNA in the ATRIP-RPA-ssDNA complex. Also, the N terminus of ATRIP associated with RPA-ssDNA in two distinct ways, indicating a dynamic and regulated association between ATRIP and RPA-ssDNA.

Quantitative trait loci analysis of heat stress resistance of spermatocytes in the MRL/MpJ mouse.

The MRL/MpJ mouse has previously been reported to possess an interesting phenotype in which spermatocytes are resistant to the abdominal temperature heat shock. In this study genetic analysis for it was performed. The phenotypes of F2 progenies produced by mating MRL/MpJ and control strain C57BL/6 mice were not segregated into two types as parental phenotypes, suggesting that the phenotype is controlled by multiple genetic loci. Thus, quantitative trait loci (QTL) analysis was performed using 98 microsatellite markers. The weight ratio of the cryptorchid testis to the intact testis (testis weight ratio) and the Sertoli cell index were used for quantitative traits. QTL analysis revealed two significant QTLs located on Chrs 1 and 11 for testis weight ratio and one significant QTL located in the same region of Chr 1 for the Sertoli cell index. A microsatellite marker locus located in the peak of the QTL on Chr 1 did not recombine with the exonuclease 1 (Exo1) gene locus in 140 F2 progenies. Mutation of the Exo1 gene was previously reported to be responsible for metaphase-specific apoptosis (MSA) of spermatocytes in the MRL/MpJ mouse. These results raise the possibility that mutation of the Exo1 gene is responsible for both MSA and heat stress resistance of spermatocytes in the MRL/MpJ mouse.

Genetic mutation associated with meiotic metaphase-specific apoptosis in MRL/MpJ mice.

It has been reported that the MRL/MpJ mouse strain shows several unique phenotypes, including rapid wound healing, inherent collagen disease, heat shock-resistant spermatocytes, and metaphase-specific apoptosis (Msa) in the testis. In the present study, we found the genetic mutation associated with Msa by chromosomal mapping with 555 backcross progeny. The Sertoli cell index of abnormal metaphasic spermatocytes was clearly divided into two groups in the first 200 male backcross progeny, which were created by mating female F1 (female C57BL/6 x male MRL/MpJ) with male MRL/MpJ mice, indicating that Msa was caused by only one gene. The result of chromosomal mapping throughout the 555 backcross progeny by using microsatellite markers and single nucleotide polymorphism (SNP) revealed that Msa was mapped on the telomeric region of chromosome 1 and was significantly linked with exonuclease 1 (Exo1) and choroideremia-like (rab escort protein 2) (Chml/Rep2) genes. It was found that the Chml/Rep2 gene was not a candidate for Msa by means of the nucleotide sequences of several inbred strains. On the Exo1 gene in strain MRL/MpJ, but not in other strains, it was surprisingly noted that the truncated forms (tr1-Exo1 and tr2-Exo1) were expressed in all tissues examined as well as normal Exo1 by reverse transcriptase-polymerase chain reaction (RT-PCR). Additionally, the truncated forms of the Exo1 gene were suggested to be transcribed by alternative splicing of the 9th exon, possibly resulting from nucleotide substitution of the branch site existing in the 8th intron. These results suggested that the testicular meiotic Msa in MRL/MpJ mice was a unique phenotype caused by incomplete alternative splicing of the Exo1 gene.

A functional truncated form of c-kit tyrosine kinase is produced specifically in the testis of the mouse but not the rat, pig, or human.

The complete nucleotide sequence of mouse-truncated mRNA of c-kit, tr-kit, has been determined using the CD1 strain. In this study, the nucleotide sequences of tr-kit from AKR/N, C57BL/6, and ICR strains of mice were determined and found to be identical, although many silent variations were found compared with the sequence in a database for CD1. Tr-kit protein consists of 12 amino acids encoded by the 16th intron and the following 190 amino acids of c-kit. In the sequences of tr-kit encoding 12 specific amino acids, no substitution was detected among the three strains and CD1. Furthermore, RT-PCR analysis clearly showed that tr-kit mRNA expression was present only in testis. No nucleotide mutation in two important regions of the presumptive promoter for tr-kit mRNA was detected within the 16th intron of the mouse strains examined. However, no functional form of tr-kit was found in the rat, pig, or human by sequence analysis and homology testing.