Biomedical subjects

Colin G Miles

Publications and source records attributed to Colin G Miles.

5 recordsLinked to original sources

PubMed2006

Expression profiling of antisense transcripts on DNA arrays.

The majority of mouse genes are estimated to undergo bidirectional transcription; however, their tissue-specific distribution patterns and physiological significance are largely unknown. This is in part due to the lack of methodology to routinely assess the expression of natural antisense transcripts (NATs) on a large scale. Here we tested whether commercial DNA arrays can be used to monitor antisense transcription in mouse kidney and brain. We took advantage of the reversely annotated oligonucleotides on the U74 mouse genome array from Affymetrix that hybridize to NATs overlapping with the sense transcript in the area of the probe set. In RNA samples from mouse kidney and brain, 11.9% and 10.1%, respectively, of 5,652 potential NATs returned positive and about half of the antisense RNAs were detected in both tissues, which was similar to the fraction of sense transcripts expressed in both tissues. Notably, we found that the majority of NATs are related to the sense transcriptome since corresponding sense transcripts were detected for 92.5% (kidney) and 74.5% (brain) of the detected antisense RNAs. Antisense RNA transcription was confirmed by real-time PCR and included additional RNA samples from heart, thymus, and liver. The randomly selected transcripts showed tissue specific expression patterns and varying sense/antisense ratios. The results indicate that antisense transcriptomes are tissue specific, and although pairing of sense/antisense transcripts are known to result in rapid degradation, our data provide proof of principle that the sensitivity of commercial DNA arrays is sufficient to assess NATs in total RNA of whole organs.

Animals↗

PubMed2003

Murine Denys-Drash syndrome: evidence of podocyte de-differentiation and systemic mediation of glomerulosclerosis.

Denys-Drash syndrome (DDS) is caused by dominant mutations of the Wilms' tumour suppressor gene, WT1, and characterized by a nephropathy involving diffuse mesangial sclerosis, male pseudohermaphroditism and/or Wilms' tumourigenesis. Previously, we reported that heterozygosity for the Wt1tmT396 mutation induces DDS in heterozygous and chimeric (Wt1tmT396/+<-->+/+) mice. In the present study, the fate of Wt1 mutant cells in chimeric kidneys was assessed by in situ marker analysis, and immunocytochemistry was used to re-examine the claim that glomerulosclerosis (GS) is caused by loss of WT1 and persistent Pax-2 expression by podocytes. Wt1 mutant cells colonized glomeruli efficiently, including podocytes, but some sclerotic glomeruli contained no detectable Wt1 mutant cells. The development of GS was preceded by widespread loss of ZO-1 signal in podocytes (even in kidneys where <5% of glomeruli contained Wt1 mutant podocytes), increased intra-renal renin expression, and de novo podocyte TGF-beta1 expression, but not podocyte Pax-2 expression or loss of WT1, synaptopodin, alpha-actinin-4 or nephrin expression. However, podocytes in partially sclerotic glomeruli that still expressed WT1 at high levels showed reduced vimentin expression, cell cycle re-entry, and re-expressed desmin, cytokeratin and Pax-2. The results suggest that: (i) GS is not due to loss of WT1 expression by podocytes; (ii) podocyte Pax-2 expression reflects re-expression rather than persistent expression, and is the consequence of GS; (iii) GS is mediated systemically and the mechanism involves activation of the renin-angiotensin system; and (iv) podocytes undergo typical maturational changes but subsequently de-differentiate and revert to an immature phenotype during disease progression.

Animals↗

PubMed2003

Faithful expression of a tagged Fugu WT1 protein from a genomic transgene in zebrafish: efficient splicing of pufferfish genes in zebrafish but not mice.

The teleost fish are widely used as model organisms in vertebrate biology. The compact genome of the pufferfish, Fugu rubripes, has proven a valuable tool in comparative genome analyses, aiding the annotation of mammalian genomes and the identification of conserved regulatory elements, whilst the zebrafish is particularly suited to genetic and developmental studies. We demonstrate that a pufferfish WT1 transgene can be expressed and spliced appropriately in transgenic zebrafish, contrasting with the situation in transgenic mice. By creating both transgenic mice and transgenic zebrafish with the same construct, we show that Fugu RNA is processed correctly in zebrafish but not in mice. Furthermore, we show for the first time that a Fugu genomic construct can produce protein in transgenic zebrafish: a full-length Fugu WT1 transgene with a C-terminal beta-galactosidase fusion is spliced and translated correctly in zebrafish, mimicking the expression of the endogenous WT1 gene. These data demonstrate that the zebrafish:Fugu system is a powerful and convenient tool for dissecting both vertebrate gene regulation and gene function in vivo.

Alternative Splicing↗

PubMed2003

Mice lacking the 68-amino-acid, mammal-specific N-terminal extension of WT1 develop normally and are fertile.

Mutations in the Wilms' tumor 1 gene, WT1, cause pediatric nephroblastoma and the severe genitourinary disorders of Frasier and Denys-Drash syndromes. High levels of WT1 expression are found in the developing kidney, uterus, and testis--consistent with this finding, the WT1 knockout mouse demonstrates that WT1 is essential for normal genitourinary development. The WT1 gene encodes multiple isoforms of a zinc finger-containing protein by a combination of alternative splicing and alternative translation initiation. The use of an upstream, alternative CUG translation initiation codon specific to mammals results in the production of WT1 protein isoforms with a 68-amino-acid N-terminal extension. To determine the function in vivo of mammal-specific WT1 isoforms containing this extension, gene targeting was employed to introduce a subtle mutation into the WT1 gene. Homozygous mutant mice show a specific absence of the CUG-initiated WT1 isoforms yet develop normally to adulthood and are fertile. Detailed histological analysis revealed normal development of the genitourinary system.

Amino Acid Sequence↗

PubMed2003

K-ras proto-oncogene exhibits tumor suppressor activity as its absence promotes tumorigenesis in murine teratomas.

Ras proteins transduce signals from membrane-bound receptors via multiple downstream effector pathways and thereby affect fundamental cellular processes, including proliferation, apoptosis, and differentiation. K-ras activating mutations play a key role in neoplastic progression and are particularly prevalent in colorectal, pancreatic, and lung cancers. The present study addressed whether the K-ras proto-oncogene displays a tumor suppressor function by comparative analysis of mouse teratomas derived from wild-type embryonic stem (ES) cells, K-ras null (K-ras(-/-)) ES cells, and K-ras(-/-) ES cells that stably reexpress either wild-type K-ras(gly12) or oncogenic K-ras(val12). K-ras(-/-) and K-ras(val12) teratomas were significantly larger than teratomas that expressed wild-type K-ras, contained significantly higher proportions of undifferentiated embryonal carcinoma-like cells, and showed significantly increased mitotic activity. However, K-ras(val12) but not K-ras(-/-) teratomas exhibited significantly higher levels of apoptosis than wild-type teratomas. K-ras(-/-) and K-ras(val12) ES cells showed a higher capacity for stem cell self-renewal in vitro compared with wild-type ES cells, and reexpression of K-ras(gly12) in K-ras(-/-) ES cells restored the K-ras(-/-) phenotype to wild-type values. Thus, in view of evidence that tumors can derive from tissue stem cells and that tumors harbor "cancer stem cells," aberrant K-ras expression could promote neoplastic progression by increasing their capacity for self-renewal.

Animals↗