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N Hastie

Publications and source records attributed to N Hastie.

17 recordsLinked to original sources
PubMed2001

Future perspectives.

Transcription is coupled to splicing and other post-transcriptional processes. The importance of transcription factors in developmental biology and disease is underlined by genetic analysis in flies and humans. The genome project is identifying large numbers of novel transcription factors and RNA-binding proteins. Proteins may have multiple functions, acting at the transcriptional and post-transcriptional levels. The vast amount of novel biological information requires new, high-throughput approaches and bioinformatics.

Animals↗
PubMed1999

Multiple roles for the Wilms' tumor suppressor, WT1.

Wilms' tumor is a childhood kidney tumor that is a striking example of the way that cancer may arise through development gone awry. A proportion of these tumors develop as a result of the loss of function mutations in the Wilms' tumor suppressor gene, WT1. Inherited mutations in the WT1 gene can lead to childhood kidney cancer, severe gonadal dysplasia, and life-threatening hypertension. Knockouts show that the gene is essential for the early stages of kidney and gonad formation. These tissues are completely absent in null mice. The WT1 gene encodes numerous protein isoforms, all of which share four zinc fingers. There is a large body of evidence supporting the notion that WT1 is a transcription factor, particularly a transcriptional repressor. Recently, however, we obtained evidence that WT1 colocalizes and is physically associated with splice factors. What is more, one alternative splice isoform of WT1 containing three amino acids, Lys-Thr-Ser (KTS; inserted between zinc fingers 3 and 4) is preferentially associated with splice factors, whereas the other alternative splice version, lacking these three amino acids, preferentially associates with the transcriptional apparatus. Both genetic and evolutionary considerations suggest that these two different forms of the protein have different functions. We will discuss recent evidence to further implicate WT1 in splicing. Our results raise the possibility that regulation of splicing is a crucial factor in the development of the genitourinary system, and that tumors may arise through aberrant splicing. To pursue the regulation and function of WT1 in whole animals, we have been introducing the human gene and large flanking regions cloned in yeast artificial chromosomes directly into mice. These studies have allowed us to dissect the function of WT1 at late as well as at early stages in organogenesis and to identify new sites and surprising new potential functions for the gene.

Alternative Splicing↗
PubMed1998

Complete sequencing of the Fugu WAGR region from WT1 to PAX6: dramatic compaction and conservation of synteny with human chromosome 11p13.

The pufferfish Fugu rubripes has a genome approximately 7.5 times smaller than that of mammals but with a similar number of genes. Although conserved synteny has been demonstrated between pufferfish and mammals across some regions of the genome, there is some controversy as to what extent Fugu will be a useful model for the human genome, e.g., [Gilley, J., Armes, N. & Fried, M. (1997) Nature (London) 385, 305-306]. We report extensive conservation of synteny between a 1.5-Mb region of human chromosome 11 and <100 kb of the Fugu genome in three overlapping cosmids. Our findings support the idea that the majority of DNA in the region of human chromosome 11p13 is intergenic. Comparative analysis of three unrelated genes with quite different roles, WT1, RCN1, and PAX6, has revealed differences in their structural evolution. Whereas the human WT1 gene can generate 16 protein isoforms via a combination of alternative splicing, RNA editing, and alternative start site usage, our data predict that Fugu WT1 is capable of generating only two isoforms. This raises the question of the extent to which the evolution of WT1 isoforms is related to the evolution of the mammalian genitourinary system. In addition, this region of the Fugu genome shows a much greater overall compaction than usual but with significant noncoding homology observed at the PAX6 locus, implying that comparative genomics has identified regulatory elements associated with this gene.

Amino Acid Sequence↗
PubMed1998

Deletion of long-range regulatory elements upstream of SOX9 causes campomelic dysplasia.

Campomelic dysplasia (CD) is a rare, neonatal human chondrodysplasia characterized by bowing of the long bones and often associated with male-to-female sex-reversal. Patients present with either heterozygous mutations in the SOX9 gene or chromosome rearrangements mapping at least 50 kb upstream of SOX9. Whereas mutations in SOX9 ORF cause haploinsufficiency, the effects of translocations 5' to SOX9 are unclear. To test whether these rearrangements also cause haploinsufficiency by altering spatial and temporal expression of SOX9, we generated mice transgenic for human SOX9-lacZ yeast artificial chromosomes containing variable amounts of DNA sequences upstream of SOX9. We show that elements necessary for SOX9 expression during skeletal development are highly conserved between mouse and human and reveal that a rearrangement upstream of SOX9, similar to those observed in CD patients, leads to a substantial reduction of SOX9 expression, particularly in chondrogenic tissues. These data demonstrate that important regulatory elements are scattered over a large region upstream of SOX9 and explain how particular aspects of the CD phenotype are caused by chromosomal rearrangements 5' to SOX9.

Animals↗
PubMed1998

Multiple roles for the Wilms' tumour suppressor gene, WT1 in genitourinary development.

Wilms' tumour is a childhood kidney cancer, and a classic example of cancer arising through disrupted development (Armstrong et al., 1992). It is one of the most common solid paediatric malignancies, affecting one in 10000 children. The genetics of Wilms' tumour is complicated, with several different genes or chromosomal regions being implicated (Armstrong et al., 1992). However, the gene we know most about is the Wilms' tumour predisposition gene, WT1 (Bickmore et al., 1992; Bruening and Pelletier, 1996). It is now clear that mutations in this gene in humans can lead to abnormalities of the kidneys and gonads, as well as to the eponymous tumour. Also, as discussed below, WT1 is essential for kidney, testis and ovary development, as revealed in knockout mice.

Animals↗
PubMed1995

DNA binding capacity of the WT1 protein is abolished by Denys-Drash syndrome WT1 point mutations.

Constitutional point mutations in the zinc finger (ZF) region of the Wilms' tumour suppressor gene 1 (WT1) lead to Denys-Drash syndrome (DDS). Patients with this syndrome display renal failure, Wilms' tumour (WT) and pseudohermaphroditism. DDS WT1 mutations fall into three major categories: (a) missense mutations altering amino acids which directly interact with the DNA target; (b) substitution of amino acids involved in zinc complexing; and (c) nonsense mutations leading to the removal of at least two zinc fingers. We have expressed the WT1 zinc fingers as glutathione-S-transferase fusion proteins, with the lysine-threonine-serine (KTS) alternate splice between ZF3 and ZF4 either present or absent. WT1 fusion constructs with all three classes of DDS mutation were also created. Wild-type and mutant fusion proteins were assayed for their DNA-binding affinity using four previously identified WT1 DNA targets: an EGR1 consensus site; murine insulin-like growth factor 2 promoter 2 (IGF2P2); a (TCC)n motif from the PDGFA-chain promoter; and +P5, a genomic fragment isolated by its affinity for WT1 + KTS. WT1-KTS bound all four targets, but WT1 + KTS only bound +P5. All three classes of DDS mutation investigated, with or without KTS, abolished binding to all four targets. This provides evidence that DDS mutations act either as dominant-negative antimorphs, or elicit their effect through disturbed isoform dosage balance.

Abnormalities, Multiple↗
PubMed1993

Frequent loss of 11p13 and 11p15 loci in male germ cell tumours.

Deletions within the short arm of the human chromosome 11 have been found to be involved in the genesis of several tumours, including different urogenital neoplasms. We have studied 31 male germ cell tumours (19 seminomas and 12 nonseminomas), and observed loss of heterozygosity at 11p loci in 40% (12/30) of these tumours [35% (9/26) at 11p13 and 31% (8/26) at 11p15]. Our data suggest that inactivation of one or more tumour suppressor genes on 11p are involved in the genesis of testicular cancer. In addition, identification of the parental origin of the allelic losses revealed a paternal loss in six patients and a maternal loss in one case.

Carcinoma in Situ↗
PubMed1992

The human PAX6 gene is mutated in two patients with aniridia.

Aniridia is an inherited ocular disorder of variable expressivity characterized by iris hypoplasia. A candidate aniridia gene, AN, which is the human homologue of the mouse Pax-6 gene, has recently been isolated by positional cloning from the WAGR region of 11p13. Here we describe mutations in this gene in two cases of sporadic aniridia, one detected at the DNA level and one at the RNA level, both of which are predicted to affect protein function. Mutations in Pax-6 have been described previously in Small eye, the proposed mouse model for aniridia. We present new phenotypic evidence for the validity of this mouse model.

Animals↗
PubMed1987

Ten families with fragile X syndrome: linkage relationships with four DNA probes from distal Xq.

We present clinical, cytogenetic, and linkage data of four DNA probes from the terminal long arm of the X chromosome in ten new families with fragile X syndrome. A prior/posterior method of multipoint linkage analysis is employed to combine these results with published data to refine the linkage map of terminal Xq. Ten possible probe/disease orderings were tested. The order with the greatest posterior probability (0.78) of the five loci is 52a-F9-fragile X gene-DX13-St14, although the order with reversal of the positions of 52a and F9 has a posterior probability 0.15. The mean estimates of the distances between the probes and the fragile X gene are 38 cM and 33 cM for the proximal probes 52a and F9, and 8 cM and 12 cM for the distal probes DX13 and St14. Although the current method of choice in the prenatal diagnosis and carrier detection of the fragile X syndrome remains detailed cytogenetic analysis, consideration is given to the potential role of these DNA probes, both singly and in pairs.

Chromosome Banding↗
PubMed1986

Use of catalase polymorphisms in the study of sporadic aniridia.

Catalase is known to map at chromosome 11p13. It is one of the closest known markers to the WAGR locus. Restriction fragment length polymorphisms (RFLP) of the catalase gene may be invaluable for studying rearrangements in somatic tumours, linkage in cases of familial Wilms tumour, and the relationship between sporadic and familial aniridia. We describe a catalase RFLP with two different enzymes and use these polymorphisms to exclude deletion of the catalase gene in patients with sporadic aniridia, including one who is known to have a deletion and another suspected of having a deletion.

Catalase↗
PubMed1985

Genetic linkage but independent expression of functional HSV-1 tk and mammalian aprt genes after cotransfer to L cells.

DNA-mediated gene transformation of mouse Ltk-aprt-hprt-cells was used to obtain stable, doubly selected transformants simultaneously expressing herpes virus thymidine kinase (TK) and mammalian adenine phosphoribosyltransferase (APRT). Cotransformants occurred at a frequency of 5 X 10(-6), a similar frequency for the transfer of the aprt marker has been previously observed. Isozyme and Southern blot analysis show that the TK and APRT expressed in these transformants resulted from gene transfer. For one stable cotransformant, [3H]thymidine [( 3H]TdR) selection against TK activity resulted in the loss of APRT activity as well, suggesting that these genes had become genetically linked together. Similarly selection against APRT expression resulted in the loss of a subset of the transferred herpes simplex virus tk genes. 5-Bromodeoxyuridine (BUdR) selected TK- variants differed from [3H]TdR selected TK- variants, in that they retained tk genes. However, BUdR-selected variants expressed full levels of APRT. Therefore, even though the transferred tk and aprt genes had become genetically linked together, they were, in this case, independently expressed since these cells were phenotypically TK- and APRT+.

Adenine Phosphoribosyltransferase↗
PubMed1984

Methylation patterns of repetitive DNA sequences in germ cells of Mus musculus.

The major and the minor satellite sequences of Mus musculus were undermethylated in both sperm and oocyte DNAs relative to the amount of undermethylation observed in adult somatic tissue DNA. This hypomethylation was specific for satellite sequences in sperm DNA. Dispersed repetitive and low copy sequences show a high degree of methylation in sperm DNA; however, a dispersed repetitive sequence was undermethylated in oocyte DNA. This finding suggests a difference in the amount of total genomic DNA methylation between sperm and oocyte DNA. The methylation levels of the minor satellite sequences did not change during spermiogenesis, and were not associated with the onset of meiosis or a specific stage in sperm development.

Animals↗
PubMedDate not supplied

Cell lineage-specific undermethylation of mouse repetitive DNA.

Several distinct cell lineages are established during mouse embryogenesis. The trophectoderm and primitive endoderm give rise to extraembryonic structures alone, while the primitive ectoderm becomes the fetus proper. Recent studies suggest that the levels of DNA modification are lower in inactive X chromosomes from extraembryonic tissues than in embryonic and adult somatic tissues. Using HpaII/MspI isoschizomers, Southern blots and cloned probes, we show here that repetitive DNA sequences from all derivatives of the two extraembryonic lineages, trophectoderm and primitive endoderm, are substantially undermethylated compared with primitive ectoderm derivatives. This contrasts with the highly methylated state of these repetitive elements observed in adult somatic tissues. Specific demethylation or inhibition of de novo methylation, or a combination of both mechanisms, may be involved. These findings suggest that elements of gene regulation dependent on DNA modification may be different in extraembryonic cell lineages.

Animals↗