Dynamic changes in the subnuclear organisation of pre-mRNA splicing proteins and RBM during human germ cell development.

RBM is a germ-cell-specific RNA-binding protein encoded by the Y chromosome in all mammals, implying an important and evolutionarily conserved (but as yet unidentified) function during male germ cell development. In order to address this function, we have developed new antibody reagents to immunolocalise RBM in the different cell types in the human testis. We find that RBM has a different expression profile from its closest homologue hnRNPG. Despite its ubiquitous expression in all transcriptionally active germ cell types, RBM has a complex and dynamic cell biology in human germ cells. The ratio of RBM distributed between punctate nuclear structures and the remainder of the nucleoplasm is dynamically modulated over the course of germ cell development. Moreover, pre-mRNA splicing components are targeted to the same punctate nuclear regions as RBM during the early stages of germ cell development but late in meiosis this spatial association breaks down. After meiosis, pre-mRNA splicing components are differentially targeted to a specific region of the nucleus. While pre-mRNA splicing components undergo profound spatial reorganisations during spermatogenesis, neither heterogeneous ribonucleoproteins nor the transcription factor Sp1 show either developmental spatial reorganisations or any specific co-localisation with RBM. These results suggest dynamic and possibly multiple functions for RBM in germ cell development.

Expression of RBM in the nuclei of human germ cells is dependent on a critical region of the Y chromosome long arm.

The association of abnormal spermatogenesis in men with Y chromosome deletions suggests that genes important for spermatogenesis have been removed from these individuals. Recently, genes encoding two putative RNA-binding proteins (RBM and DAZ/SPGY) have been mapped to two different regions of the human Y chromosome. Both of these genes encode proteins that contain a single RNA recognition motif and a (different) internally repeating sequence. Y-linked RBM homologues are found in all mammalian species. We have raised an antiserum to RBM and used it to show that RBM is a nuclear protein expressed in fetal, prepubertal, and adult male germ cells. The distribution of RBM protein in the adult correlates with the pattern of transcriptional activity in spermatogenesis, suggesting that RBM is involved in the nuclear metabolism of newly synthesized RNA. RBM sequences are found on both arms of the Y chromosome making genotype-phenotype correlations difficult for this gene family. To address the location of the functional genes and the consequences of their deletion, we examined a panel of men with Y chromosome deletions and known testicular pathologies using this antiserum. This approach enabled us to map a region of the Y chromosome essential for RBM expression. In the absence of detectable RBM expression we see stages of germ cell development up to early meiosis, but not past this point into the haploid phase of spermatogenesis.

Visualizing the spatial relationships between defined DNA sequences and the axial region of extracted metaphase chromosomes.

Using fluorescence in situ hybridization to extracted metaphase chromosomes, we present visual evidence that specific human DNA sequences occupy distinctive positions with respect to the axial region of chromosomes and that the DNA is organized into loops emanating from this region. In a stretch of unique DNA on chromosome 11, large loops of DNA can be traced and one specific region associated with the axial region of the chromosome. Within rDNA, nontranscribed spacer sequences are more closely apposed to the chromosome axis than are rRNA genes. Heterochromatic and euchromatic DNAs appear to be organized into loops of similar size. We could not detect loops at centromeres; most alphoid DNA appears to remain close to the axial region.

A high-resolution integrated physical, cytogenetic, and genetic map of human chromosome 11: distal p13 to proximal p15.1.

We describe a detailed physical map of human chromosome 11, extending from the distal part of p13 through the entirety of p14 to proximal p15.1. The primary level of mapping is based on chromosome breakpoints that divide the region into 20 intervals. At higher resolution YACs cover approximately 12 Mb of the region, and in many places overlapping cosmids are ordered in contiguous arrays. The map incorporates 18 known genes, including precise localization of the GTF2H1 gene encoding the 62-kDa subunit of TFIIH. We have also localized four expressed sequences of unknown function. The physical map incorporates genetic markers that allow relationships between physical and genetic distance to be examined, and similarly includes markers from a radiation hybrid map of 11. The cytogenetic location of cosmids has been examined on high-resolution banded chromosomes by fluorescence in situ hybridization, and FLpter values have been determined. The map therefore fully integrates physical, genic, genetic, and cytogenetic information and should provide a robust framework for the rapid and accurate assignment of new markers at a high level of resolution in this region of 11p.

Organization of the region encompassing the human serum amyloid A (SAA) gene family on chromosome 11p15.1.

The four members of the human serum amyloid A protein (SAA) gene family are clustered on human chromosome 11p15.1. Three genes are differentially expressed and encode small apolipoproteins of M(r) 12-19 kDa: SAA1 and SAA2 encode the acute phase SAAs (A-SAAs), and SAA4 encodes the constitutively expressed SAA (C-SAA). A fourth locus, SAA3, is a pseudogene. The human SAA gene family encompasses approximately 150 kb contained on a 900-kb yeast artificial chromosome contig. SAA1 and SAA2 are 15-20 kb apart and are arranged in divergent transcriptional orientations. SAA4 is 9 kb downstream of SAA2 and in the same orientation. SAA3 is 110 kb downstream of SAA4, and its relative orientation could not be determined. All genes known to be in the same human and mouse syntenic linkage group as SAA were mapped within the contig. Interphase FISH was used to orientate the region relative to the centromere: cen-LDHC-LDHA-SAA1-SAA2-SAA4-SAA3-TP H-D11S18- KCNC1-MYOD1-pter.

Colocalization of the human CD59 gene to 11p13 with the MIC11 cell surface antigen.

The human CD59 gene encodes a cell surface antigen detected by MEM43 and other antibodies. It has homology to the mouse Ly-6 genes that map on mouse chromosome 15 and are involved in lymphocyte signal transduction. CD59 may play a role in protecting against complement-mediated lysis. The human CD59 gene had been previously localized to 11p13-p14. We expected, on the basis of synteny arguments, that CD59 would map in 11p14. However, we have precisely localized the human CD59 gene to band p13 of chromosome 11 by somatic cell genetics and by pulsed-field gel electrophoresis; indeed the gene is often deleted in WAGR individuals. This region of chromosome 11 is syntenic with mouse chromosome 2. This suggests that CD59 is not a homolog of the mouse Ly-6 genes on mouse chromosome 15, but rather is a related gene. The human CD59 gene is shown to map within 500 kb of another cell surface marker, MIC11, the gene for which has not been cloned. This localization and the results of immunoprecipitation experiments suggest that the CD59 gene could encode the MIC11 antigen; alternatively this region of 11p13 may contain a cluster of genes encoding cell surface molecules.

Modulation of DNA binding specificity by alternative splicing of the Wilms tumor wt1 gene transcript.

The technique of whole-genome polymerase chain reaction was used to study the DNA binding properties of the product of the wt1 gene. The zinc finger region of this gene is alternatively spliced such that the major transcript encodes a protein with three extra amino acids between the third and fourth fingers. The minor form of the protein binds specifically to DNA. It is now shown that the major form of wt1 messenger RNA encodes a protein that binds to DNA with a specificity that differs from that of the minor form. Therefore, alternative splicing within the DNA binding domain of a transcription factor can generate proteins with distinct DNA binding specificities and probably different physiological targets.