Carvedilol Suppresses Apoptosis and Ion Channel Remodelling of HL-1 Cardiac Myocytes Expressing E334K cMyBPC.

BACKGROUND: Besides its antiarrhythmic action, carvedilol has an activity to suppress cardiac tissue damage. However, it is unknown whether it has any effect on cellular apoptosis and ion channel remodelling. PURPOSE: To know whether carvedilol has any effect on apoptosis and ion channel remodeling of HL-1 cells expressing E334K MyBPC, and comparing it with bisoprolol. METHOD: We examined effects of carvedilol and bisoprolol on the levels of pro- and anti-apoptotic proteins and ion channels as well as apoptosis of HL-1 cells transfected with E334K MyBPC using Western blot and flow cytometry. RESULTS: Carvedilol decreased the protein levels of p53, Bax and cytochrome c and increased that of Bcl-2 in HL-1 cells expressing E334K MyBPC. Bisoprolol failed to affect the protein levels. Both carvedilol and bisoprolol increased the protein levels of Cav1.2 but not that of Nav1.5. Carvedilol was stronger than bisoprolol at decreasing the number of annexin-V positive cells in HL-1 cells expressing E334K MyBPC. CONCLUSION: Carvedilol suppressed apoptosis of HL-1 cells expressing E334K MyBPC through modification of pro- and anti-apoptotic proteins, whose was associated with an increase of Cav 1.2 protein expression.

Simultaneous treatment with azelnidipine and olmesartan inhibits apoptosis of Hl-1 cardiac myocytes expressing E334k cMyBPC.

BACKGROUND: Apoptosis appears to play an important role in the pathogenesis of hypertrophic cardiomyopathy (HCM). We have previously reported 3 HCM patients carrying the E334K MYBPC3, and that heterologous expression of E334K cMyBPC in cultured cells induced apoptosis. The purpose of this study was to identify pharmacological agents that would inhibit apoptosis in HL-1 cardiomyocytes expressing E334K cMyBPC. METHODS AND RESULTS: E334K cMyBPC expression in cells increased levels of pro-apoptosis (p53, Bax and cytochrome c) and decreased levels of anti-apoptosis (Bcl-2 and Bcl-XL). While the beta blocker carvedilol (1 µM) normalized the level of p53 and Bcl-2 and the calcium channel blocker (CCB) bepridil (0.5 µM) normalized that of Bcl-2, both the CCB azelnidipine (1 µM) and the angiotensin receptor blocker (ARB) olmesartan (10 µM) normalized those of p53, Bax, cytochrome c, and Bcl-XL. Among those proteins, cytochrome c was the one which showed the highest degree of change. Both azelnidipine (0.1 µM) and olmesartan (1 µM) reduced the level of cytochrome c by 40.2 ± 4.3% and 31.3 ± 5.1%, respectively. The CCB amlodipine and the ARB valsartan reduced it only by 19.1 ± 2.1% and 20.1 ± 5.2%, respectively. Flow cytometric analysis and annexin V staining showed that treatment of cells with azelnidipine (0.1 µM) plus olmesartan (0.3 µM) or that with amlodipine (0.1 µM) plus valsartan (0.3 µM) reduced the number of apoptotic cells by 35.8 ± 10.5% and 18.4 ± 3.2%, respectively. CONCLUSION: Azelnidipine plus olmesartan or amlodipine plus valsartan inhibited apoptosis of HL-1 cells expressing E334K cMyBPC, and the former combination was more effective than the latter.

Transcriptional activation of the anchoring protein SAP97 by heat shock factor (HSF)-1 stabilizes K(v) 1.5 channels in HL-1 cells.

BACKGROUND AND PURPOSE: The expression of voltage-dependent K(+) channels (K(v) ) 1.5 is regulated by members of the heat shock protein (Hsp) family. We examined whether the heat shock transcription factor 1 (HSF-1) and its inducer geranylgeranylacetone (GGA) could affect the expression of K(v) 1.5 channels and its anchoring protein, synapse associated protein 97 (SAP97). EXPERIMENTAL APPROACH: Transfected mouse atrial cardiomyocytes (HL-1 cells) and COS7 cells were subjected to luciferase reporter gene assay and whole-cell patch clamp. Protein and mRNA extracts were subjected to Western blot and quantitative real-time polymerase chain reaction. KEY RESULTS: Heat shock of HL-1 cells induced expression of Hsp70, HSF-1, SAP97 and K(v) 1.5 proteins. These effects were reproduced by wild-type HSF-1. Both heat shock and expression of HSF-1, but not the R71G mutant, increased the SAP97 mRNA level. Small interfering RNA (siRNA) against SAP97 abolished HSF-1-induced increase of K(v) 1.5 and SAP97 proteins. A luciferase reporter gene assay revealed that the SAP97 promoter region (from -919 to -740) that contains heat shock elements (HSEs) was required for this induction. Suppression of SIRT1 function either by nicotinamide or siRNA decreased the level of SAP97 mRNA. SIRT1 activation by resveratrol had opposing effects. A treatment of the cells with GGA increased the level of SAP97 mRNA, K(v) 1.5 proteins and I(Kur) current, which could be modified with either resveratrol or nicotinamide. CONCLUSIONS AND IMPLICATIONS: HSF-1 induced transcription of SAP97 through SIRT1-dependent interaction with HSEs; the increase in SAP97 resulted in stabilization of K(v)1.5 channels. These effects were mimicked by GGA.

Refined human artificial chromosome vectors for gene therapy and animal transgenesis.

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis.

Correction of a genetic defect in multipotent germline stem cells using a human artificial chromosome.

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including regulatory elements. Multipotent germline stem (mGS) cells have a great potential for gene therapy because they can be generated from an individual's testes, and when reintroduced can contribute to the specialized function of any tissue. As a proof of concept, we herein report the functional restoration of a genetic deficiency in mouse p53-/- mGS cells, using a HAC with a genomic human p53 gene introduced via microcell-mediated chromosome transfer. The p53 phenotypes of gene regulation and radiation sensitivity were complemented by introducing the p53-HAC and the cells differentiated into several different tissue types in vivo and in vitro. Therefore, the combination of using mGS cells with HACs provides a new tool for gene and cell therapies. The next step is to demonstrate functional restoration using animal models for future gene therapy.

Proteomic signatures and aberrations of mouse embryonic stem cells containing a single human chromosome 21 in neuronal differentiation: an in vitro model of Down syndrome.

Neurodegeneration in fetal development of Down syndrome (DS) patients is proposed to result in apparent neuropathological abnormalities and to contribute to the phenotypic characteristics of mental retardation and premature development of Alzheimer disease. In order to identify the aberrant and specific genes involved in the early differentiation of DS neurons, we have utilized an in vitro neuronal differentiation system of mouse ES cells containing a single human chromosome 21 (TT2F/hChr21) with TT2F parental ES cells as a control. The paired protein extracts from TT2F and TT2F/hChr21 cells at several stages of neuronal differentiation were subjected to two-dimensional polyacrylamide gel electrophoresis protein separation followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to identify the proteins differentially expressed between TT2F and TT2F/hChr21 cells. We provide here a novel set of specific gene products altered in early differentiating DS neuronal cells, which differs from that identified in adult or fetal brain with DS. The aberrant protein expression in early differentiating neurons, due to the hChr21 gene dosage effects or chromosomal imbalance, may affect neuronal outgrowth, proliferation and differentiation, producing developmental abnormalities in neural patterning, which eventually leads to formation of a suboptimal functioning neuronal network in DS.

A new mouse model for Down syndrome.

Trisomy 21 (Ts21) is the most common live-born human aneuploidy and results in a constellation of features known as Down syndrome (DS). Ts21 is a frequent cause of congenital heart defects and the leading genetic cause of mental retardation. Although overexpression of a gene(s) or gene cluster on human chromosome 21 (Chr 21) or the genome imbalance by Ts21 has been suggested to play a key role in bringing about the diverse DS phenotypes, little is known about the molecular mechanisms underlying the various phenotypes associated with DS. Four approaches have been used to model DS to investigate the gene dosage effects of an extra copy of Chr 21 on various phenotypes; 1) Transgenic mice overexpressing a single gene from Chr 21, 2) YAC/BAC/PAC transgenic mice containing a single gene or genes on Chr 21, 3) Mice with intact/partial trisomy 16, a region with homology to human Chr 21 and 4) Human Chr 21 transchromosomal (Tc) mice. Here we review our new model system for the study of DS using the Tc technology, including the biological effects of an additional Chr 21 in vivo and in vitro.

Tumour invasion and metastasis are promoted in mice deficient in tenascin-X.

BACKGROUND: Tenascin-X (TNX) is a member of the tenascin family of large oligomeric glycoproteins of the extracellular matrix (ECM). To determine whether TNX plays a part in tumour invasion and metastasis and to disclose its normal physiological role, we disrupted its gene in mouse embryonic stem cells by homologous recombination and created mice deficient in TNX. RESULTS: TNX-null mutant (TNX-/-) mice arose at normal frequency and showed no obvious defects during their adult life. However, when TNX-/- mice were subcutaneously inoculated in foot-pads with a highly invasive and metastatic cell line, B16-BL6 melanoma cells, the primary tumour size at 30 days after inoculation in the TNX-/- mice had increased by 1.2-fold compared with that in wild-type mice, and the invasion to the ankle and pulmonary metastasis in TNX-/- mice were also augmented by 2.2-fold and 6.8-fold, respectively, compared to those in wild-type mice. To disclose the molecular mechanism(s) of the promotion of tumour invasion and metastasis in TNX-/- mice, we measured the protein levels of matrix metalloproteinases (MMPs), which are recognized as playing a key role in these events, in the foot-pad homogenates of TNX-/- mice prior to the inoculation of melanoma cells. Gelatin zymography showed that the activities of proMMP-2, active MMP-2 and proMMP-9 were significantly higher in TNX-/- mice than in wild-type mice. Furthermore, a Northern blot analysis demonstrated that this increased activity of MMP-2 in TNX-/- mice was due to the induced expression of MMP-2 at the transcriptional level. The elevated expression of MMP-2 and MMP-9 resulted in decreased laminin levels, to less than half that of wild-type mice in the homogenates of TNX-/- mice. CONCLUSIONS: TNX deficiency led to an increase in the production of MMPs, and the increased activity of MMPs may result in the degradation of laminin. Consequently, the melanoma cells inoculated in TNX-/- mice might facilitate invasion and metastasis. These results imply that TNX is required for impeding the invasion and metastasis of tumour cells.

A novel in vitro system for analyzing parental allele-specific histone acetylation in genomic imprinting.

One of the obstacles in studying human genomic imprinting is distinguishing the parental origin of alleles in diploid cells. To solve this problem, we have constructed a library of mouse A9 hybrids in which individual clones contain a single human chromosome of known parental origin. Here we extend this in vitro system to the analysis of the role of histone acetylation in the allelic expression of human imprinted genes. The levels of histone H4 acetylation of the imprinted human LIT1, H19, and SNRPN genes were examined by a chromatin immunoprecipitation (ChIP) assay in mouse A9 hybrids with a single human chromosome of known parental origin. We demonstrated that H4 histones associated with the actively expressed alleles of imprinted LIT1, H19, and SNRPN genes were highly acetylated, whereas they were hypoacetylated in the silent alleles. Furthermore, treatment of A9 hybrids with trichostatin A (TSA), an inhibitor of histone deacetylase, resulted in transcriptional reactivation of the silent alleles for LIT1 and SNRPN, suggesting that histone deacetylation is one of the key regulatory mechanisms in genomic imprinting. These results indicate that our monochromosomal hybrid system is a new technology for analyzing histone modifications between parental alleles in human imprinted genes.

Epigenetic silencing of PEG3 gene expression in human glioma cell lines.

Genomic imprinting, the phenomenon in which alleles of genes are expressed differentially depending on their parental origins, has important consequences for mammalian development, and disturbance of normal imprinting leads to abnormal embryogenesis and some inherited diseases and is also associated with various cancers. In the context of screening for novel imprinted genes on human chromosome 19q13.4 with mouse A9 hybrids, we identified a maternal allele-specific methylated CpG island in exon 1 of paternally expressed imprinted gene 3 (PEG3), a gene that exhibits paternal allele-specific expression. Because PEG3 expression is downregulated in some gliomas and glioma cell lines, despite high-level expression in normal brain tissues, we investigated whether the loss of PEG3 expression is related to epigenetic modifications involving DNA methylation. We found monoallelic expression of PEG3 in all normal brain tissues examined and five of nine glioma cell lines that had both unmethylated and methylated alleles; the remaining four glioma cell lines exhibited gain of imprinting with hypermethylated alleles. In addition, treatment of glioma cell lines with the DNA demethylating agent 5-aza-2'-deoxycytidine reversed the silencing of PEG3 biallelically. In this article, we report that the epigenetic silencing of PEG3 expression in glioma cell lines depends on aberrant DNA methylation of an exonic CpG island, suggesting that PEG3 contributes to glioma carcinogenesis in certain cases.

Targeted disruption of the human LIT1 locus defines a putative imprinting control element playing an essential role in Beckwith-Wiedemann syndrome.

Human chromosome 11p15.5 harbors an intriguing imprinted gene cluster of 1 Mb. This imprinted domain is implicated in a wide variety of malignancies and Beckwith-Wiedemann syndrome (BWS). Recently, several lines of evidence have suggested that the BWS-associated imprinting cluster consists of separate chromosomal domains. We have previously identified LIT1, a paternally expressed antisense RNA within the KvLQT1 locus through a positional screening approach using human monochromosomal hybrids. KvLQT1 encompasses the translocation breakpoint cluster in BWS and patients exhibit frequent loss of maternal methylation at the LIT1 CpG island, implying a regulatory role for the LIT1 locus in coordinate control of the imprinting cluster. Here we generated modified human chromosomes carrying a targeted deletion of the LIT1 CpG island using recombination-proficient chicken DT40 cells. Consistent with the prediction, this mutation abolished LIT1 expression on the paternal chromosome, accompanied by activation of the normally silent paternal alleles of multiple imprinted loci at the centromeric domain including KvLQT1 and p57(KIP2). The deletion had no effect on imprinting of H19 located at the telomeric end of the cluster. Our findings demonstrate that the LIT1 CpG island can act as a negative regulator in cis for coordinate imprinting at the centromeric domain, thereby suggesting a role for the LIT1 locus in a BWS pathway leading to functional inactivation of p57(KIP2). Thus, the targeting and precise modification of human chromosomal alleles using the DT40 cell shuttle system can be used to define regulatory elements that confer long-range control of gene activity within chromosomal domains.

The ActR-I activin receptor protein is expressed in notochord, lens placode and pituitary primordium cells in the mouse embryo.

ActR-I is a type I serine/threonine kinase receptor which has been shown to bind activin and bone morphogenetic proteins (BMPs). To study the function of ActR-I, we have generated novel monoclonal antibodies that specifically recognize the extracellular domain of mouse ActR-I. We examined the level of ActR-I protein during mouse development by immunohistochemistry. We found that in the embryonic body, ActR-I protein first appears in a restricted part of the primitive streak region and is present throughout the length of notochord. Furthermore, ActR-I protein is expressed in the facial sensory organ primordia, including eye area, otic vesicle and olfactory placode, which all contain invaginating ectoderm. In addition, ActR-I is produced in pituitary primordium (Rathke's pouch), mammary buds and the epithelial layer of branchial arches. Interestingly, in the lens placodes and in early Rathke's pouch, ActR-I protein is transiently localized at the apical surface of the epithelial cells, indicating the presence of an apical-basal asymmetry in these cells.

Cloning of inv, a gene that controls left/right asymmetry and kidney development.

Most vertebrate internal organs show a distinctive left/right asymmetry. The inv (inversion of embryonic turning) mutation in mice was created previously by random insertional mutagenesis; it produces both a constant reversal of left/right polarity (situs inversus) and cyst formation in the kidneys. Asymmetric expression patterns of the genes nodal and lefty are reversed in the inv mutant, indicating that inv may act early in left/right determination. Here we identify a new gene located at the inv locus. The encoded protein contains 15 consecutive repeats of an Ank/Swi6 motif at its amino terminus. Expression of the gene is the highest in the kidneys and liver among adult tissues, and is seen in presomite-stage embryos. Analysis of the transgenic genome and the structure of the candidate gene indicate that the candidate gene is the only gene that is disrupted in inv mutants. Transgenic introduction of a minigene encoding the candidate protein restores normal left/right asymmetry and kidney development in the inv mutant, confirming the identity of the candidate gene.

Proto-oncogene of int-3, a mouse Notch homologue, is expressed in endothelial cells during early embryogenesis.

BACKGROUND: Notch and its homologues are key regulatory receptors of the cell fate decision in various developmental processes. The int-3 oncogene was originally identified as a frequent target in Mouse Mammary Tumour Virus (MMTV)-induced mammary tumours and has been regarded as a Notch homologue, based on its similarity to the intracellular domain of Notch. Studies with int-3 transgenic mice have suggested that the int-3 transgene affects the differentiation capacity of stem cells and leads to neoplastic proliferation in epithelial cells. However, the exact nature and the in vivo expression pattern of the int-3 proto-oncogene are unknown. The function of gene products in embryogenesis is also not clear. RESULTS: We isolated cDNA clones corresponding to the proto-oncogene of int-3 and analysed its overall structure. The predicted amino acid sequence of the int-3 proto-oncogene contains the conserved motif found in Notch family receptors. Therefore, we name Notch-4 for the int-3 proto-oncogene. However, Notch-4 has fewer EGF repeats and shows less similarity to Notch, compared with other mammalian Notch homologues. In embryogenesis, the expression of Notch-4 was detected in endothelial cells of blood vessels forming tissues such as the dorsal aorta, intersegmental vessels, yolk sac vessels, cephalic vessels, heart, vessels in branchial arches, and capillary plexuses. In these tissues, Notch-4 expression coincided with flk-1, the major regulatory gene of vasculogenesis and angiogenesis. We also found that Notch-4 expression was up-regulated in vitro during the differentiation of endothelial cells from embryonic stem cells (ES cells). CONCLUSION: The endothelial cell specific expression pattern of Notch-4, as well as its structural similarity of Notch, suggest that Notch-4 is an endothelial cell specific homologue of Notch and it may play a crucial role in vasculogenesis and angiogenesis.

Gene transfection of mouse primordial germ cells in vitro and analysis of their survival and growth control.

We evaluated electroporation, liposome-mediated transfection, and the calcium phosphate (CaPO4) coprecipitation method for gene transfection of mouse primordial germ cells (PGCs) in culture as a prelude to the investigation of molecular mechanisms of the germ cell development. We found that electroporation severely damaged PGCs, and the efficiency of liposome-mediated transfection was very low. In contrast, using the CaPO4 coprecipitation method, 18% of PGCs transfected with plasmid pSV-LT expressed simian virus 40 large tumor antigen (SV 40 T-Ag) transiently. However, we did not detect any effects on the proliferation and survival of PGCs obtained from the embryonic gonads at 11.5 days postcoitum (d.p.c.) during 2 days of culture after the transfection. PGCs isolated from the 11.5-d.p.c. gonads change from spread- to round-shape and exhibit growth arrest during a few days of culture, and these rounded PGCs quickly disappear from the culture. We found that the transfection and expression of Bcl-XL or adenovirus type 2 E1B 19,000-molecular-weight protein (E1B 19K) significantly promoted the survival of PGCs and retarded the disappearance of rounded PGCs from the culture system. These results suggest that the Bcl-XL or E1B 19K can prevent the apoptosis of PGCs and inhibit the cell death of the rounded PGCs in culture.

Functional conservation of mouse Notch receptor family members.

All the known members of the mouse Notch receptor family were examined for their biochemical function by interaction with a DNA binding protein RBP-Jkappa. mNotch2, mNotch3 and int3 (= mNotch4) were shown to interact with RBP-Jkappa by the GST-fusion pull down assay and dominant negative competition with Epstein Barr virus nuclear antigen 2. Furthermore the intracellular region of int3 was shown to transactivate the Epstein Barr virus TP1 promoter. These results indicate that all mouse Notch family members have biochemical functions similar to mNotch1, which transduces proliferative signal by direct interaction with the DNA binding protein RBP-Jkappa.

Functional requirement of gp130-mediated signaling for growth and survival of mouse primordial germ cells in vitro and derivation of embryonic germ (EG) cells.

Leukemia inhibitory factor (LIF) is a cytokine known to influence proliferation and/or survival of mouse primordial germ cells (PGC) in culture. The receptor complex for LIF comprises LIF-binding subunit and non-binding signal transducer, gp130. The gp130 was originally identified as a signal-transducing subunit of interleukin (IL)-6 and later also found to be a functional component of receptor complexes for other LIF-related cytokines (oncostatin M [OSM], ciliary neurotrophic factor [CNTF] and IL-11). In this study, we have analyzed the functional role of gp130-mediated signaling in PGC growth in vitro. OSM was able to fully substitute for LIF; both cytokines promoted the proliferation of migratory PGC (mPGC) and enhanced the viability of postmigratory (colonizing) PGC (cPGC) when cultured on SI/SI4-m220 cells. Interestingly, IL-11 stimulated mPGC growth comparable to LIF and OSM, but did not affect cPGC survival. IL-6 and CNTF did not affect PGC. In addition, a combination of IL-6 and soluble IL-6 binding subunit (sIL-6R), which is known to activate intracellular signaling via gp130, fully reproduced the LIF action of PGC. Both in the presence and absence of LIF, addition of neutralizing antibody against gp130 in culture remarkably blocked cPGC survival. These results suggest a pivotal role of gp130 in PGC development, especially that it is indispensable for cPGC survival as comparable to the c-KIT-mediated action. We have further demonstrated that a combination of LIF with forskolin or retinoic acid, a potent mitogen for PGC, supported the proliferation of PGC, leading to propagation of the embryonic stem cell-like cells, termed embryonic germ (EG) cells. Since EG cells were also obtained by using OSM or the IL-6/sIL-6R complex in place of LIF, a significant contribution of gp130-mediated signaling in EG cell formation was further suggested.

Autonomous regulation of proliferation and growth arrest in mouse primordial germ cells studied by mixed and clonal cultures.

In culture, mouse primordial germ cells (PGCs) proliferate and undergo growth arrest with a time course similar to that in vivo. It is unclear whether this behavior is regulated autonomously or by coexisting somatic cells. We performed mixed culture experiments using PGCs from 8.5- and 11.5-d.p.c. embryos and found no interaction between the PGCs and somatic cells at the two stages. Next, we carried out clonal culture of PGCs and examined the proliferation of and morphological change in individual clones. Such clonal culture did not reveal any subpopulation of PGCs with an increased growth rate or less differentiated characteristics, which might have been suggested by formation of the embryonic germ cell lines. Our results suggest that there is an autonomous regulation of growth and cell shape change in PGCs which occur as stochastical events but are not strictly timed by the number of cell divisions.

Recombination in the class III region of the mouse major histocompatibility complex.

The sites of meiotic recombination in the class II region of the mouse major histocompatibility complex (MHC) are clustered at hotspots. To search for hotspots in the class III region, we mapped recombinational breakpoints of 79 Ab:H2-D recombinants with 11 DNA markers; these included Tnx, the gene for an extracellular matrix protein, tenascin X, the Notch-related Int3 gene, and a microsatellite marker, D17Mit13, none of which had previously been mapped precisely. The results gave the gene order of Eb-61.1-Int3-Tnx-Cyp21/C4-Bf-Hsp68c-D17Mit13+ ++-Tnfa/Tnfb-D. The crossover sites in 40 of the 79 recombinants were confined within the Eb/Int3:Tnx/Cyp21 interval. The result demonstrated that an unequal distribution of recombination is a general feature of the mouse MHC, suggesting the presence of a recombinational hotspot within the Int3:Tnx interval.