RBM Gene Research Articles

Transcriptomic Response of Differentiating Porcine Myotubes to Thermal Stress and Donor Piglet Age.

Climate change is a current concern that directly and indirectly affects agriculture, especially the livestock sector. Neonatal piglets have a limited thermoregulatory capacity and are particularly stressed by ambient temperatures outside their optimal physiological range, which has a major impact on their survival rate. In this study, we focused on the effects of thermal stress (35 °C, 39 °C, and 41 °C compared to 37 °C) on differentiating myotubes derived from the satellite cells of Musculus rhomboideus, isolated from two different developmental stages of thermolabile 5-day-old (p5) and thermostable 20-day-old piglets (p20). Analysis revealed statistically significant differential expression genes (DEGs) between the different cultivation temperatures, with a higher number of genes responding to cold treatment. These DEGs were involved in the macromolecule degradation and actin kinase cytoskeleton categories and were observed at lower temperatures (35 °C), whereas at higher temperatures (39 °C and 41 °C), the protein transport system, endoplasmic reticulum system, and ATP activity were more pronounced. Gene expression profiling of HSP and RBM gene families, which are commonly associated with cold and heat responses, exhibited a pattern dependent on temperature variability. Moreover, thermal stress exhibited an inhibitory effect on cell cycle, with a more pronounced downregulation during cold stress driven by ADGR genes. Additionally, our analysis revealed DEGs from donors with an undeveloped thermoregulation capacity (p5) and those with a fully developed thermoregulation capacity (p20) under various cultivation temperature. The highest number of DEGs and significant GO terms was observed under temperatures of 35 °C and 37 °C. In particular, under 35 °C, the DEGs were enriched in insulin, thyroid hormone, and calcium signaling pathways. This result suggests that the different thermoregulatory capacities of the donor piglets determined the ability of the primary muscle cell culture to differentiate into myotubes at different temperatures. This work sheds new light on the underlying molecular mechanisms that govern piglet differentiating myotube response to thermal stress and can be leveraged to develop effective thermal management strategies to enhance skeletal muscle growth.

A novel phase variant of the cholera pathogen shows stress-adaptive cryptic transcriptomic signatures.

BackgroundIn a process known as phase variation, the marine bacterium and cholera pathogen Vibrio cholerae alternately expresses smooth or rugose colonial phenotypes, the latter being associated with advanced biofilm architecture and greater resistance to ecological stress. To define phase variation at the transcriptomic level in pandemic V. cholerae O1 El Tor strain N16961, we compared the RNA-seq-derived transcriptomes among the smooth parent N16961, its rugose derivative (N16961R) and a smooth form obtained directly from the rugose at high frequencies consistent with phase variation (N16961SD).ResultsDifferentially regulated genes which clustered into co-expression groups were identified for specific cellular functions, including acetate metabolism, gluconeogenesis, and anaerobic respiration, suggesting an important link between these processes and biofilm formation in this species. Principal component analysis separated the transcriptome of N16961SD from the other phase variants. Although N16961SD was defective in biofilm formation, transcription of its biofilm-related vps and rbm gene clusters was nevertheless elevated as judged by both RNA-seq and RT-qPCR analyses. This transcriptome signature was shared with N16961R, as were others involving two-component signal transduction, chemotaxis, and c-di-GMP synthesis functions.ConclusionsPrecise turnarounds in gene expression did not accompany reversible phase transitions (i.e., smooth to rugose to smooth) in the cholera pathogen. Transcriptomic signatures consisting of up-regulated genes involved in biofilm formation, environmental sensing and persistence, chemotaxis, and signal transduction, which were shared by N16961R and N16961SD variants, may implicate a stress adaptation in the pathogen that facilitates transition of the N16961SD smooth form back to rugosity should environmental conditions dictate.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3233-x) contains supplementary material, which is available to authorized users.

Structural Insights into RbmA, a Biofilm Scaffolding Protein of V. Cholerae

V. cholerae can form sessile biofilms associated with abiotic surfaces, cyanobacteria, zoo-plankton, mollusks, or crustaceans. Along with the vibrio polysaccharide, secreted proteins of the rbm gene cluster are key to the biofilm ultrastructure. Here we provide a thorough structural characterization of RbmA, a protein involved in mediating cell-cell and cell-biofilm contacts. We correlate our structural findings with initial ligand specificity screening results, NMR protein-ligand interaction analysis, and complement our results with a full biocomputational study.

Effects of diethylhexyl phthalate (DEHP) given neonatally on spermatogenesis of mice

We previously demonstrated that the effects of diethylhexyl phthalate (DEHP) alter reproduction function on male mice. Immature male mice were treated daily with DEHP from postnatal day 7-21, 7-35, 7-49, in a dose-dependent manner. As results, both the quality and quantity of spermatozoa were decreased in 60-day-old mice. The results by RT-PCR analysis indicated that DDx3Y, Usp9Y, RBM, E1F1AY, EGF, FSHR and EGFR genes were down-regulated, and LHR, Cyp17a1 and Cyp19a1 were down-regulated in response to DEHP. These genes were selected based on their markedly increased or decreased expression levels. However, DEHP had no effect on the meiotic process and recombination levels in male mouse germ cells. Treatment with DEHP induced histopathological changes in the testes. Taken together, these results provide a new insight into the molecular mechanisms underlying the detrimental impacts of DEHP in humans and wildlife.

AZF Gene Expression Analysis in Peripheral Leukocytes and Testicular Cells from Idiopathic Infertility

The aim of this study was to assess the frequency of AZF microdeletions in peripheral leukocytes and testicular cells in Chinese men with idiopathic infertility. Expression in testicular cells was also determined. In this study, we screened 62 idiopathic infertile patients, in whom karyotype, sperm count and hormonal parameters were evaluated. Genomic DNA was extracted from the peripheral leukocytes. Molecular analysis was performed by two multiplex polymerase chain reactions (PCR) using a set of eight sequence tagged sites (STS) from 3 different regions of the Y chromosome. Total cellular RNA was extracted from the testicular tissue using a Trizol-method. Reverse Transcription (RT) reactions were performed to synthesize cDNA. Amplification of DFFRY, RBM and DAZ genes was performed to analyze their expression in testicular cells. In this cohort, we found 12 submicroscopic deletions (12/62, 19.4%). Nine patients (9/33, 27.2%) were detected in the azoospermic group and three (3/29, 10.3%) in the severe oligozoospermic group. RT-PCR analysis from testicular cells gave normal amplifications for SRY and DFFRY mRNA in 62 idiopathic patients; two patients were negative for RBM expression; no RBM and DAZ were detected for a case; 12 patients had no expression in the AZFc region involving the DAZ gene. Of 12 cases, three patients with normal PCR analysis of DAZ gene on genomic DNA showed no RT-PCR amplification for DAZ mRNA. The use of RT-PCR of specific spermatid expressed genes in conjunction with examining microdeletions using peripheral leukocytes is suggested to avoid the transmission of the Y chromosomal microdeletions from a father to a son via testicular sperm aspiration (TESE), intracytoplasmic sperm injection (JCSI).

Positive correlation between the expression of X‐chromosome RBM genes (RBMX, RBM3, RBM10) and the proapoptotic Bax gene in human breast cancer

In a recent report, it has been postulated that the ubiquitous RBM proteins might constitute a novel family of apoptosis modulators. We measured the expression of the X-chromosome RBM genes (RBMX, RBM3, and RBM10) in 122 breast cancers by means of differential RT-PCR. Using the same method, we also studied the expression of the apoptosis-related genes Bcl-2 and Bax. Markers of hormone dependence (estrogen and progesterone receptors), proliferation (Ki67 and DNA-ploidy), angiogenesis (VEGF and CD105), as well as oncogene (c-erb-B2), and tumor suppressor gene (p53) expression were also analyzed. The expression of all X-chromosome RBM genes was significantly associated with the expression of the proapoptotic Bax gene (RBMX, P=0.039; RBM3, P<0.001; RBM10 large variant, P<0.001; RBM10 small variant, P<0.001). Furthermore, the expression of both RBM10 variants was significantly associated with the expression of the VEGF gene (large variant, P=0.004; small variant, P=0.003). We also found an association of borderline significance (P=0.05) between the expression of RBM3, the large variant of RBM10 and wild-type p53. Expression of the small RBM10 variant, finally, was associated with high proliferation of the tumors (Ki67>or=20%; P=0.037). The expression of both RBM10 variants seems to be interdependent to a significant degree (r=0.26, P=0.006). From these results, it seems that the X-chromosome, through its RBM genes, plays a formerly unknown role in the regulation of programmed cell death (apoptosis) in breast cancer.

The role of potential splicing factors including RBMY, RBMX, hnRNPG‐T and STAR proteins in spermatogenesis*

Investigations into the RBM gene family are uncovering networks of protein interactions which regulate RNA processing, and which might operate downstream of signal transduction pathways. Similar pathways likely operate in germ cells and somatic cells, with RBMY, hnRNPGT and T-STAR proteins providing germ cell-specific components. These pathways may be important for normal germ cell development, and might be compromised in men with Y chromosome deletions affecting RBMY gene expression. The STAR proteins have multiple functions in pre-mRNA splicing, signalling and cell cycle control. These processes might have to be very finely regulated during germ cell development, which involves both two sequential meiotic divisions (meiosis I and II) as well as mitotic (spermatogonial) cell divisions, and which is controlled by paracrine signalling within the testis from Sertoli cells.

Y-chromosome microdeletion and phenotype in cytogenetically normal men with idiopathic azoospermia

Objective: To determine the prevalence of microdeletions of the long arm of chromosome Y within the AZFa, AZFb, and AZFc subregions in patients with idiopathic azoospermia, and then correlate the microdeletions with clinical phenotypes to determine the most important subregion for screening. Design: Controlled clinical study. Setting: Male infertility clinic, Kobe University Hospital. Patient(s): Among 89 consecutive azoospermic patients, those whose infertility was related to known hereditary, endocrine, or obstructive causes or a cytogenetic abnormality were excluded; 54 remaining patients were studied using a polymerase chain reaction (PCR). Of these patients, 33 had Sertoli cell only syndrome, 10 had maturation arrest, and 11 had hypospermatogenesis. Intevention(s): Blood and semen samples and testicular biopsies were obtained from all of the participants. Main Outcome Measure(s): We performed semen analysis, polymerase chain amplification of 28 DNA loci on the long arm of the Y chromosome involving the DAZ (deleted in azoospermia), and measured the plasma FSH, LH, testosterone, prolactin, and estradiol levels. Result(s): Microdeletions were detected in 14 of the 54 patients (nine with Sertoli cell only, three with maturation arrest, and two with hypospermatogenesis). Most microdeletions involved AZFb or AZFc. Patients with hypospermatogenesis or maturation arrest showed deletion only in AZFc. The DAZ gene was deleted in four patients with Sertoli cell only and one patient with maturation arrest. The RBM gene was deleted in two patients with Sertoli cell only who had particularly large deletions, but in no patients with arrest or hypospermatogenesis. Conclusion(s): Cytogenetically azoospermic patients should be examined for microdeletions before undertaking assisted reproduction. AZFc may be the most important subregion to screen. In addition, intact AZFa and AZFb subregions may be important for the presence of germ cells.

Y-chromosome microdeletion and its effect on reproductive decisions in Taiwanese patients presenting with nonobstructive azoospermia

Objectives. To investigate the position, extent, and frequency of Y chromosome microdeletions in Taiwanese patients presenting with nonobstructive azoospermia, and to investigate the effect of microdeletions on reproductive decisions. Methods. We studied 176 consecutive men with azoospermia in our urology clinic. Polymerase chain reaction tests were performed in 94 patients with nonobstructive azoospermia, and a series of 27 sequence-tagged sites (STSs) mapped within intervals 5 and 6 of Yq11 was selected for analysis. Clinical genetics counseling was provided to couples with microdeletions, and these couples made their own choices about further treatment modalities. Results. Among 94 patients screened for microdeletion, 11 (11.7%) showed microdeletions of one or more STSs. One had a deletion confined to the azoospermia factor b (AZFb) region (encompassing the RBM gene). Two were found to have deletions of both the AZFb and AZFc regions. Eight patients had deletions in the AZFc region (encompassing the DAZ gene). Five had deletions distal to the DAZ gene family. One had multiple, noncontiguous deletions. In 8 patients with testicular histology available, a lack of genotype/phenotype correlation was noted. Of the 11 couples with deletions, 3 thought microdeletion was a serious defect and opted for an artificial insemination of donor or adoption, 5 chose intracytoplasmic sperm injection, and the other 3 decided to undergo treatment with Chinese medicinal herbs. Conclusions. The most commonly deleted region in the Taiwanese population is AZFc. The genes implicated in Taiwanese spermatogenesis defects are the DAZ and RBM gene families. Twenty-seven percent of couples with microdeletions deferred assisted reproductive technologies because of concern about their underlying genetic defects.

A High Frequency of Y Chromosome Deletions in Males With Nonidiopathic Infertility

Microdeletions of the long arm of the human Y chromosome are associated with spermatogenic failure and have been used to define three regions of Yq (AZFa, AZFb, and AZFc) that are recurrently deleted in infertile males. In a blind study we screened 131 infertile males (46 idiopathic and 85 nonidiopathic) for Y chromosome microdeletions. Nineteen percent of idiopathic males, with an apparently normal 46,XY chromosome complement had microdeletions of either the AZFa, AZFb, or AZFc region. There was no strict correlation between the extent or location of the deletion and the phenotype. The AZFb deletions did not include the active RBM gene. Significantly, a high frequency of microdeletions (7%) was found in patients with known causes of infertility and a 46,XY chromosome complement. These included deletions of the AZFb and AZFc regions, with no significant difference in the location or extent of the deletion compared with the former group. It is recommended that all males with reduced or absence sperm counts seeking assisted reproductive technologies be screened for deletions of the Y chromosome.

Absence of Y chromosome microdeletions involving the DAZ (deleted in azoospermia) and RBM (ribonucleic acid-binding motif) genes in patients with chordee without hypospadias.

The presence or absence of 33 DNA loci was investigated with a focus on the long arm of Y chromosome (Yq) interval 6 involving the DAZ and RBM genes, by means of a polymerase chain reaction strategy, in 10 patients with hypospadias. All patients did not show any of the 33 DNA loci. It would appear that chordee without hypospadias does not involve interval 6 of the Y chromosome.

Mice with Y chromosome deletion and reduced Rbm genes on a heterozygous Dazl1 null background mimic a human azoospermic factor phenotype.

A subset of azoospermia or oligozoospermia patients have microdeletions in defined regions of their Y chromosome, namely the AZFa, b, and c regions. Candidate genes in humans that may cause the azoospermia factor (AZF) phenotype have been assigned to these regions and can include the DAZ and RBM genes. Part of the variability in the AZFc phenotype might be due to interaction between the effects of deleting the DAZ and RBM genes. We mimicked human deletions of RBM and DAZ in the mouse by crossing male mice with a deleted Y chromosome with a reduced number of Rbm genes (Y(d1)) to heterozygote Dazl1 null female mice to study the interaction of the Dazl1 and Rbm or other genes located in the Y(d1) deletion interval. Dazl-/+ Y(d1) animals showed a significant reduction in the sperm count (P < 0.001), an increase of abnormal sperm heads and prominent mid-piece defects of the tails compared to either mutation alone (P < 0.001). Hence, Dazl1 and the genes removed on the Y(d1) chromosome are active in different pathways contributing to different stages of spermatogenesis. Reduction of Dazl1 and Rbm genes as well as/or deletion of the Y chromosome in mice gives rise to a phenotype similar to the heterogeneous AZFc phenotype observed in humans.

A high frequency of Y chromosome deletions in males with nonidiopathic infertility.

Microdeletions of the long arm of the human Y chromosome are associated with spermatogenic failure and have been used to define three regions of Yq (AZFa, AZFb, and AZFc) that are recurrently deleted in infertile males. In a blind study we screened 131 infertile males (46 idiopathic and 85 nonidiopathic) for Y chromosome microdeletions. Nineteen percent of idiopathic males, with an apparently normal 46,XY chromosome complement had microdeletions of either the AZFa, AZFb, or AZFc region. There was no strict correlation between the extent or location of the deletion and the phenotype. The AZFb deletions did not include the active RBM gene. Significantly, a high frequency of microdeletions (7%) was found in patients with known causes of infertility and a 46,XY chromosome complement. These included deletions of the AZFb and AZFc regions, with no significant difference in the location or extent of the deletion compared with the former group. It is recommended that all males with reduced or absence sperm counts seeking assisted reproductive technologies be screened for deletions of the Y chromosome.

Absence of testicular DAZ gene expression in idiopathic severe testiculopathies.

Deletions of the DAZ (deleted in azoospermia) gene family are frequently responsible for male infertility and are generally assessed by analyses of genomic DNA extracted from peripheral leukocytes. The multicopy nature of this gene prevents the distinction of intragenic deletions or deletions not involving the whole DAZ gene cluster. Thus it is still unclear whether each DAZ copy is effectively expressed in the testis. We analysed, by reverse transcription-polymerase chain reaction (RT-PCR), the expression of DAZ, RBM and SRY genes, in testicular cells from infertile men affected by idiopathic severe hypospermatogenesis, obstructive azoospermia and Sertoli cell-only syndrome. Normal mRNA for DAZ, RBM and SRY were observed in obstructive azoospermia, whereas only SRY transcripts were detected when only Sertoli cells were present. Nine out of 10 patients affected by idiopathic severe hypospermatogenesis had normal expression of SRY, RBM and DAZ, while in one patient no DAZ transcript was detected, suggesting that his testiculopathy was related to the absence of DAZ expression. The lack of DAZ mRNA in testicular cells with an apparently normal DAZ gene constitution on DNA extracted from leukocytes may be explained by different hypotheses: (i) not all the copies of the DAZ gene cluster are transcribed in the germ cells and the reported patient had a small deletion involving only the active ones; (ii) the patient may be mosaic for the DAZ gene having a normal constitution in leukocytes and be deleted for DAZ gene in the testis; (iii) abnormalities of DAZ transcription may exist. These findings highlight the intrinsic interpretative difficulties of normal PCR analysis for DAZ and RBM on leukocytes and suggest caution in the use of germ cells for assisted reproductive techniques in these cases to avoid transmission of genetic abnormalities to male offspring.

Human male infertility and Y chromosome deletions: role of the AZF-candidate genes DAZ, RBM and DFFRY.

Microdeletions in Yq11 overlapping three distinct 'azoospermia factors' (AZFa-c) represent the aetiological factor of 10-15% of idiopathic azoospermia and severe oligozoospermia, with higher prevalence in more severe testiculopathies, such as Sertoli cell-only syndrome. Using a PCR-based screening, we analysed Yq microdeletions in 180 infertile patients affected by idiopathic Sertoli cell-only syndrome and different degrees of hypospermatogenesis, compared with 50 patients with known causes of testicular alteration, 30 with obstructive azoospermia, and 100 normal fertile men. In idiopathic severe testiculopathies (Sertoli cell-only syndrome and severe hypospermatogenesis), a high prevalence of microdeletions (34.5% and 24.7% respectively) was found, while milder forms were not associated with Yq alteration. No deletions were found in testiculopathies of known aetiology, obstructive azoospermia, normal fertile men and male relatives of patients with deletions. Deletions in the AZFc region involving the DAZ gene were the most frequent finding and they were more often observed in severe hypospermatogenesis than in Sertoli cell-only syndrome, suggesting that deletions of this region are not sufficient to cause complete loss of the spermatogenic line. Deletions in AZFb involving the RBM gene were less frequently detected and there was no correlation with testicular phenotype, with an apparent minor role for such gene in spermatogenesis. The DFFRY gene was absent in a fraction of patients, making it a candidate AZFa gene. Our data suggest that larger deletions involving more than one AZF-candidate gene are associated with a more severe testicular phenotype.

CDNA Cloning and Characterization of PD1: A Novel Human Testicular Protein with Different Expressions in Various Testiculopathies

A novel human cDNA sequence has been isolated from human testis cDNA library. This sequence, named PD1, reveals an open reading frame encoding a protein of 520 amino acids. A partial sequence similarity has been found with the RBM gene involved in the regulation of human spermatogenesis. Northern blot analysis for PD1 mRNA from several human tissues demonstrated two distinct transcripts of 2.7 (more abundant) and 4.0 kb and revealed that PD1 is expressed in testis and to a lesser extent also in spleen, thymus, and prostate. Immunohistochemical analysis of human testis showed that this protein is detected in the cytoplasm of Sertoli cells. Antibodies against a rhPD1 fragment were used for Western blot analysis, which confirmed the presence of a 60-kDa molecule in crude extract of human testicular cells obtained from fine-needle aspiration and showed different patterns in various testiculopathies, suggesting a role for such gene in human spermatogenesis.

Cromossoma Y e infertilidade masculina.

Spermatogenesis is a complex physiological process characterized by an orderly proliferation and differentiation of germ cell types, from diploid spermatogonial stem cells to haploid spermatids, and after spermiogenesis to spermatozoa. It is known that male reproductive capacity is deficient in about one half of infertile couples. Effective treatment is available only for a small fraction of these infertile men. In most cases, the cause of male infertility is unknown. Aetiologically, male infertility may result from genetic and non-genetic causes. Among the genetic factors known to disturb spermatogenesis, chromosomal aberrations, involving autosomes and/or sex chromosomes, are well known. However, molecular genetic causes of idiopathic male infertility have been accumulating in the last years, predominantly for the Y chromosome. Localization of genes that control spermatogenesis on Yq11 was first proposed, on cytogenetic evidence, by Tiepolo and Zuffardi in 1976. Since then, many subsequent reports using molecular genetics methods have supported the initial proposal by detecting submicroscopic interstitial deletions on Yq11, present in patients with idiophatic azoospermia and a cytogenetically normal Y chromosome. Recently, four spermatogenesis candidate Y-linked genes (or gene families), RBM, DAZ, SPGY and TSPY have been cloned and characterized. In the euchromatic Y chromosome long arm three intervals--AZFa, AZFb and AZFc--were also defined. All of them may contain genes necessary for normal spermatogenesis. All the four genes have a testis-specific expression and three of them (RBM, DAZ and SPGY) code for ribonucleoproteins with a single RNA recognition motif. Candidate genes with in AZFb are some members of the RBM gene family and within AZFc are the DAZ and SPGY gene family. While AZFc deletions are associated with azoospermia or with severe oligoteratoasthenozoospermia, microdeletions involving AZFa or AZFb cause azoospermia only.

Short stature and azoospermia in a patient with Y chromosome long arm deletion

We report on a 42-year old male with short stature, azoospermia and a wide deletion of long arm of Y chromosome. On physical examination, the patient showed height of 149 cm (< 1 degree centile) and reduced volume (3 ml) and consistency of the testes. On hormonal evaluation, he showed increased serum gonadotropins and normal serum testosterone levels though its HCG stimulated levels were limited. Serum thyroid hormones were normal. Serum GH levels in baseline evaluation as well as after GHRH and GHRH + pyridostigmine administration were normal. Serum IGF I levels were lower than normal in baseline evaluation whereas its response to the GH administration was in the normal range. The bilateral testicular biopsy showed tubular atrophy, hyalinosis, interstitial sclerosis and a histological picture of a Sertoli cell only syndrome. Moreover the patient showed arthropathy, otopathy, small chin, small mouth and truncal obesity. On genetic evaluation, the patient showed a 46,X,delY (pter--q11.1:) karyotype and loss of several DNA loci on Yq. In fact he preserved short arm SRY, centromeric DYZ3 and more proximal euchromatic region Yq loci, including DYS270, DYS271, DYS272, DYS11, DYS273, DYS274, DYS148, DYS275, and missed more distal DNA loci from DYS246 to DYZ2. These results disclosed a wide Y long arm deletion, including all hypothized Yq azoospermia loci (except for AZFa and probably for one of the RBM genes, which lie proximally to the deletion) and possibly the Y-specific growth control region (GCY), mapped between DYS11 and DYS246 loci. This deletion is responsible for the complete azoospermia of the patient and probably also for his short stature, even if other factors could be implicated in the statural impairment. It further possibly allowed to relate the GCY gene(s) to the control of GH or IGF-I receptor or post-receptor pathway, being the alteration of this gene(s) consistent with the hormonal pattern of the patient.

Part of the RBM gene cluster is located distally to the DAZ gene cluster in human Yq11.23.

Normal human Y and inverted Y chromosomes were chosen for physical fluorescence in situ hybridization (FISH) mapping of RBM and DAZ probes for the relative positioning of the RBM and DAZ gene clusters in interval 6 of the human Y chromosome. The inversion breakpoint in Yq11.23 turned out to be distal to the DAZ gene cluster, as the entire DAZ signal appears in the short arm of the inv(Y) chromosome. On the contrary, this inversion breakpoint in Yq11.23 divides the RBM signal cluster, leaving a weaker signal on the long arm while bringing the main RBM signal to the short arm of the inv(Y) chromosome. Thus, it can be concluded that, in contrast to previous claims, part of the RBM gene cluster is located distally to the DAZ gene cluster in deletion interval 6 of the human Y chromosome.

High-resolution fluorescence in situ hybridization of human Y-linked genes on released chromatin.

Genes within the differential region of the human Y chromosome do not recombine, and therefore the determination of their location depends on physical mapping. Yeast artificial chromosome (YAC) contigs spanning the euchromatic region of the human Y have become a powerful tool for the generation of an overlapping clone map. With this approach, however, complete physical mapping is difficult in Y euchromatic regions that are rich in repetitive sequences. We have, therefore, made use of the fluorescence in situ hybridization technique as an alternative strategy for physically mapping the PRKY and AMELY genes as well as the TSPY, RBM and DAZ gene families to human Y chromosomes in prometaphase and to extended Y chromatin in interphase. From our results, the following order of gene sequences in interval 3 of the short arm of the human Y chromosome is suggested: TSPY major with few RBM sequences interspersed-PRKY-AMELY-TSPY minor with few RBM sequences interspersed-cen. On the long arm, RBM sequences appear to be distributed over wide regions of intervals 5 and 6 with few TSPY sequences interspersed. Distal to an RBM signal cluster, a large cluster of DAZ signals is located with only a few DAZ and RBM signals overlapping in between the two clusters.