Search PubMed⌕ Search

Biomedical subjects

R A McGowan

Publications and source records attributed to R A McGowan.

17 recordsLinked to original sources

Identification and isolation of a BTB-POZ-containing gene expressed in oocytes and early embryos of the zebrafish Danio rerio.

In this report, we describe the cloning of a cDNA from the zebrafish Danio rerio encoding a protein containing a BTB-POZ domain closely resembling the BTBD1 and BTBD2 proteins previously identified in mammals. However, unlike other BTB-POZ-containing genes, expression of this gene in adults is most abundant in oocytes, where the RNA can be detected at all stages of oogenesis examined. The presence of the RNA persists through early cleavage, but is decreased significantly by gastrulation. Although the function of this gene has yet to be determined, its resemblance to the BTB-POZ family of genes coupled with its expression pattern suggests that it may have an important function in oogenesis and (or) early zebrafish development.

Amino Acid Sequence↗

Global changes in genomic methylation levels during early development of the zebrafish embryo.

We have examined the methylation status of the zebrafish genome during early embryogenesis and we find evidence that methylation fluxes do occur in that organism. The parental genetic contributions to the zygote are, initially, differently methylated with the genome of the sperm being hypermethylated relative to the genome of the oocyte. Post-fertilization there is an immediate decrease in methylation of the embryonic genome but the methylation begins to increase rapidly and is re-established by the gastrulation stage. These results are consistent with the results of Santos et al. (Dev Biol 241:172-182, 2002), who examined the methylation of early mouse embryos, and this conservation argues that demethylation/re-methylation is an important part of vertebrate development.

Animals↗

Cloning and sequence analysis of a zebrafish cDNA encoding DNA (cytosine-5)-methyltransferase-1.

The zebrafish has become a well-established animal model for the analysis of development and of several disease phenotypes. Several of the favorable traits that make it a popular model organism would also be beneficial for the study of normal and abnormal vertebrate development in which DNA methylation may play a role. We report the determination of the full-length cDNA sequence corresponding to the zebrafish DNA (cytosine-5-) methyltransferase gene, Dnmt1. It is 4,907 bases long and has an open reading frame predicted to encode a 1,499 amino acid protein that is similar in size and sequence to a number of other methyltransferases identified in other organisms.

Amino Acid Motifs↗

DNA methylation and genome imprinting in the zebrafish, Danio rerio: some evolutionary ramifications.

Although methylation has been recognized as an important component in a number of developmental processes in mammals, in zebrafish almost nothing is known about this epigenetic modification. This is despite the fact that the zebrafish is becoming increasingly popular as a developmental model system. The little work that has been done on methylation and development in fish concerns genomic imprinting. In mammals, imprinting results in an inability to reproduce parthenogenetically because a genetic contribution from both parents is necessary to successfully complete development. However, this is not true of zebrafish, and a number of the theories that have been presented to explain the evolution of imprinting are not consistent with imprinting in these fish. A new model is presented that discusses some of the potential evolutionary ramifications of methylation and imprinting and that leads to the suggestion that imprinting may actually be a simple genetic mechanism to enhance the efficient evolution of both individual genetic loci and combinations of loci with related functions, without risking the population as a whole. This model can accommodate all of the information known about imprinting, including its broad phylogenetic range, imprinting by both males and females, and the diverse nature of the genes that are known to be imprinted.

Animals↗

Expression of murine renin genes during fetal development.

Fetuses were examined to produce a developmental profile of renin expression in the kidneys and adrenal glands in single renin gene and two renin gene strains of mice. Sites of renin expression were detected by in situ hybridization using an 35S-labeled antisense RNA probe complimentary to the renin cDNA. Accumulation of renin transcripts in the adrenal gland reached a maximum at 15.5 days post coitum for all strains examined, but declined to undetectable levels by birth in one gene strains, while in two gene strains, the levels of renin transcripts lessened and by birth became limited to the developing inner cortex. Kidney renin transcripts were first detected at 14.5 days post coitum in the newly developing arteries in fetuses of both genotypes of mice. As the renal arterial tree developed, renin mRNA containing cells were progressively localized to more distal blood vessels and finally to the specialized cells of the afferent arteriole (juxtaglomerular cells). These results were confirmed by examining the localization of immunoreactive T antigen in transgenic fetuses. These mice carried a transgene which placed the SV40 T antigen structural gene under control of renin regulatory elements. Expression of T antigen occurred at the same sites in the kidneys and adrenal glands as renin mRNA. Furthermore, in strains with two renin genes, primer extension analysis indicated transcripts from both genes were present in equal proportion in combined kidney and adrenal gland extracts of total RNA. These transcripts were full length in size. The transient localization of renin mRNA in cells of the fetal intrarenal arteries is consistent with the notion that renin may be a useful marker for the developing renal vasculature.

Adrenal Glands↗

Expression of the DBA/2J Ren-2 gene in the adrenal gland of transgenic mice.

To characterize further the tissue-specific control of the mouse Ren-2 gene, and in particular its expression in the adrenal gland, we have introduced the DBA/2 Ren-2 gene into the genome of Ren-1c/Ren-1c mice. Here we report our observations on Ren-2 transgenic mice. Expression was found in the correct spectrum of tissues and included appropriate hormonal control in the submandibular gland. Quantitatively transcript levels varied both positively (adrenal gland and sex-accessory tissue) and negatively (submandibular gland and kidney) with respect to normal Ren-2 expression. In the DBA/2 inbred mouse strain expression in the female adrenal gland cycles during oestrus between the X-zone and the zona fasciculata. Transgene expression within the adrenal gland was restricted to the X-zone. Therefore this phenotype, which is characteristic of most two-renin-gene strains of mice, contrasts with that found in the strain DBA/2 from which the transgene was derived. This suggests that cell-specific expression in the DBA/2 adrenal gland is mediated in trans by at least one additional locus. We demonstrate that suitable genetic crosses of the transgenic mice can partially restore the cycling phenotype.

Adrenal Glands↗

Allele-specific expression of the murine Ren-1 genes.

Inbred strains of mice carry either one of two alleles for the Ren-1 gene on chromosome 1; Ren-1c or Ren-1d. The Ren-1d allele is found in association with a tightly linked duplicated locus, Ren-2. The relative accumulations of renin transcripts derived from each locus were found to differ between mice with one or two renin loci in three extra renal tissues, the adrenal gland, testis, and sex accessory gland tissue. We find that the two Ren-1 alleles exhibit characteristic expression patterns in these tissues. Using genetic analyses and an allele-specific dideoxynucleotide primer extension assay we have further characterized the expression differences in these tissues. The results suggest that the allele-specific expression patterns observed for the Ren-1 genes are regulated by closely associated sequences in cis.

Adrenal Glands↗

Effect of androgen and thyroid hormones on renin-1 messenger ribonucleic acid levels in mouse submandibular gland.

The synthesis of renin and other biologically active polypeptides in the granular convoluted tubule cells of the mouse submandibular gland (SMG) is regulated by androgen and thyroid hormones. In this study genetically hypothyroid (hyt/hyt) mice carrying a single renin structural gene (Ren-1) were used to investigate the mechanism of hormonal action in mouse SMG. Treatment of female mice with 5 alpha-dihydrotestosterone (DHT) and/or thyroxine (T4) enhanced renin-1 activity and increased renin-1 mRNA, determined by Northern analysis. Compared to euthyroid (hyt/+) littermates, hyt/hyt mice had lower basal levels of renin-1 mRNA and a blunted response to either hormone alone. DHT and T4 acted synergistically to increase renin-1 activity and renin-1 mRNA in the SMG of hyt/hyt females. Furthermore, levels of renin-1 activity and renin-1 mRNA varied concordantly in the SMG of these animals. These data indicate that androgen and thyroid hormones influence levels of renin-1 in mouse SMG primarily by regulating the amount of renin-1 mRNA available for translation.

Animals↗

An integrated approach to board development.

Growing demands on hospital boards call for systematic efforts to increase the effectiveness of individual board members. These activities must go beyond the provision of traditional education programs.

Education, Continuing↗

Modulation of kidney renin messenger RNA levels during experimentally induced hypertension.

Several experimental procedures produce dramatic alterations in kidney renin production, leading to increased plasma renin levels with attendant hypertension in animal model systems. The pattern of changes for kidney renin messenger RNA (mRNA) levels relative to changes for tissue and plasma renin activity was studied in Sprague-Dawley rats made hypertensive by either coarctation of the aorta between the two renal arteries or clipping of the left renal artery. In both models, the renin mRNA content of the contralateral hypertrophied kidney transiently decreased to undetectable levels while the ischemic kidney exhibited transient increases in renin mRNA. In aorta-coarctated rats ischemic kidney renin mRNA increased 10-fold to 16-fold during the first 3 days after coarctation but returned to the level observed in sham-operated rats 14 days after operation. However, differences between the time course and magnitude of changes in renin mRNA levels and the pattern of alteration in tissue and plasma renin activities were observed. Thus, although the kidney renin mRNA transiently increased in hypertensive animals, the extent of this increase was insufficient to account for the 30-fold to 50-fold increases in plasma renin activity. Similarly, the transient increase in kidney renin mRNA was inconsistent with only a twofold increase in tissue renin enzyme activity of the ischemic kidney. These data indicate that in addition to alterations in the kidney renin mRNA pool, posttranslational processing and/or release of renin from the kidney are cocontributors in regulating the plasma renin levels in these experimental models.

Animals↗

Basal forebrain innervation of rodent neocortex: studies using acetylcholinesterase histochemistry, Golgi and lesion strategies.

Acetylcholinesterase (AChE)-rich projections from basal forebrain to neocortex cerebri were characterized in the present study. The purpose was to investigate 3 aspects of these projections in rats and mice that have been incompletely described in previous work: intracortical organization of the fibers, subcortical pathways and axonal branching patterns of individual basal forebrain neurons. AChE histochemistry, lesions and Golgi impregnations were the principal strategies employed in this light microscopic study. The moderately dense, AChE-stained innervation of neocortex can be altered by intracortical lesions. The results depended on the region involved and the orientation of the lesion. Sagittal knife cuts had barely detectable effects, regardless of sites. Coronal knife cut lesions in medial cortex resulted in substantial loss of staining in cingulate and medial occipital fields. In contrast, coronal lesions of lateral or anterior cortex produce only small zonal reductions in staining. The interpretation of the latter findings that we favor is that AChE-rich basal forebrain fibers enter lateral/anterior cortex and branch densely there, but in tangentially limited and overlapping terminal domains. Observations on the topography and targets of AChE-rich basal forebrain cortical afferents revealed that the fibers could be grouped based on certain characteristics. Three sets of fibers were distinguishable: anterior pathway innervating cortex of the frontal pole. These fibers were traceable to the region of the substantia innominata/nucleus basalis. They crossed the neostriatum and external capsule in the sagittal plane, forming in 3 dimensions an orderly sheet-like array of fibers bridging the anteroventral surface of the neostriatum with nearby polar cortex medial pathway innervating cingulate and medial occipital cortex. Emerging predominantly from the region of the diagonal band, the fibers run caudally as a triangular bundle in deep layer VI of cingulate cortex. lateral pathway innervating most of remaining lateral neocortex. The fibers radiate out from substantia innominata/nucleus basalis with a complex 3-dimensional organization. In all pathways, fibers enter and initially run within layer VI before ascending pialward, although the intracortical course in layer VI differs between pathways. These fibers primarily terminate in layer V with a secondary concentration in layer I. However, the latter appears to receive substantial AChE-stained inputs from other sources, possibly intracortical, as well. The pathways overlap at their respective boundary zones. This system is comparably organized in rats and mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholinesterase↗

Expression of tissue-specific Ren-1 and Ren-2 genes of mice: comparative analysis of 5'-proximal flanking regions.

All inbred strains of mice carry the Ren-1 structural gene, which encodes the renin-1 isozyme, the classical renin activity found in kidneys. In addition, some strains carry a second renin structural gene, Ren-2, which encodes the predominantly expressed submaxillary gland renin isozyme, renin-2. Ren-1 and Ren-2 exhibit markedly different patterns of tissue-specific expression. In an effort to understand the molecular basis for this differential expression, detailed analysis of the genomic sequences corresponding to the Ren-1 and Ren-2 genes, and the transcripts originating from these loci, was undertaken. Sequence analysis of regions proximal to the structural genes indicated the presence of eucaryotic consensus sequences for transcription. These sequence motifs were strongly conserved between Ren-1 and Ren-2. Approximately 150 bases upstream from the major transcription initiation site, significant differences between these genes were apparent, including the presence of a repetitive DNA element in the Ren-2 copy as well as other breaks in homology and sequence curiosities. Strong homology between Ren-1 and Ren-2 resumed at a point ca. 200 bases further upstream on Ren-1. S1 analysis of submaxillary gland and kidney RNA populations indicated that the majority of transcripts initiate at homologous positions on Ren-1 and Ren-2. On a per cell basis, the accumulation of Ren-1 transcripts in the kidney and Ren-2 transcripts in the submaxillary gland are probably equivalent. These results suggest that it is tissue-specific utilization of the homologous start sites that is critical to their differential patterns of expression. Models which can account for this observation are presented. Interestingly, we found a minor fraction of transcripts initiating 5' to the major transcription start site. These transcripts encoded an open reading frame which may add an additional 23 amino acids to the N-terminus of the renin precursor.

Animals↗

Tissue and gene specificity of mouse renin expression.

The Ren-1 locus of mice encodes the protease renin, which with converting enzyme processes angiotensinogen to the potent vasopressor angiotensin II. Some strains of mice appear to carry a duplication of the renin structural gene (Ren-2) near the Ren-1 locus. Strains with the gene duplication can exhibit as much as 100-fold higher levels of submaxillary gland renin compared to strains with a single gene copy. In contrast, kidney renin levels appear to be unaffected by the gene duplication. Sequence analysis of a 319 bp renin cDNA recombinant isolated from a kidney library from the two-gene strain DBA/2Ha corresponds to a transcript of the Ren-1 gene. Moreover, a single base substitution of A for G at residue #996 in the kidney renin mRNA creates a potential glycosylation recognition site that may, in part, account for the differential glycosylation of kidney and submaxillary gland renins. In addition, our tissue surveys indicate that mature mRNAs from the Ren loci are detectable in adrenal gland and testes, as well as sublingual and parotid salivary glands, and reveal length variation for the renin transcripts in at least the submaxillary gland.

Animals↗