Michael Clinton

Dosage compensation in birds.

The Z and W sex chromosomes of birds have evolved independently from the mammalian X and Y chromosomes [1]. Unlike mammals, female birds are heterogametic (ZW), while males are homogametic (ZZ). Therefore male birds, like female mammals, carry a double dose of sex-linked genes relative to the other sex. Other animals with nonhomologous sex chromosomes possess dosage compensation systems to equalize the expression of sex-linked genes. Dosage compensation occurs in animals as diverse as mammals, insects, and nematodes, although the mechanisms involved differ profoundly [2]. In birds, however, it is widely accepted that dosage compensation does not occur [3-5], and the differential expression of Z-linked genes has been suggested to underlie the avian sex-determination mechanism [6]. Here we show equivalent expression of at least six of nine Z chromosome genes in male and female chick embryos by using real-time quantitative PCR [7]. Only the Z-linked ScII gene, whose ortholog in Caenorhabditis elegans plays a crucial role in dosage compensation [8], escapes compensation by this assay. Our results imply that the majority of Z-linked genes in the chicken are dosage compensated.

Sexing chick embryos: a rapid and simple protocol.

1. Analysis of gene expression in the developing chick gonads requires the collection of male and female tissues from embryos between 3.5 d and 8.5 d of development. However, male and female chick embryos are indistinguishable by morphological examination before d 7.5 of development. 2. Sex identification of earlier embryos is only possible by molecular methods, which at present are laborious and time consuming. 3. We have devised a PCR-based sexing protocol which combines both sex specific and control reactions in a single tube assay. The assay is rapid and effective over a wide range of DNA concentrations and is tolerant of poor quality DNA. 4. Procedures are described for identifying the sex of individual embryos using either tissue samples or a small number of cells recovered from amniotic fluid.

An overview of factors influencing sex determination and gonadal development in birds

Abstract. The morphological development of the embryonic gonads is very similar in birds and mammals, and recent evidence suggests that the genes involved in this process are conserved between these classes of vertebrates. The genetic mechanism by which sex is determined in birds remains to be elucidated, although recent studies have reinforced the contention that steroids may play an important role in the structural development of the testes and ovaries in birds. So far, few genes have been assigned to the avian sex chromosomes, but it is known that the Z and W chromosomes do not share significant homology with the mammalian X and Y chromosomes. The commercial importance of poultry breeding has motivated considerable investment in developing physical and genetic maps of the chicken genome. These efforts, in combination with modern molecular approaches to analyzing gene expression, should help to elucidate the sex-determining mechanism in birds in the near future.

Genetic complexity of an obesity QTL (Fob3) revealed by detailed genetic mapping.

Obesity is proving to be a serious health concern in the developed world as well as an unwanted component of growth in livestock production. While recent advances in genetics have identified a number of monogenic causes of obesity, these are responsible for only a small proportion of human cases of obesity. By divergent selection for high and low fat content over 60 generations, we have created Fat (F) and Lean (L) lines of mice that represent a model of polygenic obesity similar to the situation in human populations. From previous crosses of these lines, four body fat quantitative trait loci (QTL) were identified. We have created congenic lines (Fchr15L), by recurrent marker-assisted backcrossing, to introgress the QTL region with the highest LOD score, Fob3 on Chr 15, from the L-Iine into the F-line background. We have further mapped this QTL by progeny testing of recombinants, produced from crosses between the F-line and congenic Fchrl5L mice, showing that the Fob3 QTL region is a composite of at least two smaller effect QTL—the proximal QTL Fob3a is a late-onset obesity QTL, whereas the distal Fob3b is an early-onset obesity QTL.

Overexpression of Raidd cDNA inhibits differentiation of mouse preadipocytes

Abstract. RAIDD (RIP‐associated ICH‐1 homologous protein with a death domain) is an adaptor molecule that mediates the action of cysteine proteases involved in apoptosis. To study the possibility of a novel system of cell ablation mediated by RAIDD, a preadipocyte cell line (3T3L1) was stably transfected with a plasmid containing the murine Raidd cDNA under the control of the adipocyte specific promoter aP2. Instead of the expected apoptosis, a blockage to differentiation upon hormonal induction was observed as judged by an absence of lipid accumulation, a lack of expression of adipocyte‐specific genes and a fibroblastic appearance. Proliferation rate of Raidd‐transfected clones remained unaffected. Overexpression of Raidd cDNA in 3T3L1 cell therefore inhibited differentiation, suggesting that Raidd plays a role in controlling differentiation of mouse preadipocytes and, perhaps, in other cell types, in addition to its established role in apoptosis.

Gonadotropin-Releasing Hormone Regulates Expression of the DNA Damage Repair Gene, Fanconi anemia A, in Pituitary Gonadotroph Cells

Abstract Gonadal function is critically dependant on regulated secretion of the gonadotropin hormones from anterior pituitary gonadotroph cells. Gonadotropin biosynthesis and release is triggered by the binding of hypothalamic GnRH to GnRH receptor expressed on the gonadotroph cell surface. The repertoire of regulatory molecules involved in this process are still being defined. We used the mouse LβT2 gonadotroph cell line, which expresses both gonadotropin hormones, as a model to investigate GnRH regulation of gene expression and differential display reverse transcription-polymerase chain reaction (RT-PCR) to identify and isolate hormonally induced changes. This approach identified Fanconi anemia a (Fanca), a gene implicated in DNA damage repair, as a differentially expressed transcript. Mutations in Fanca account for the majority of cases of Fanconi anemia (FA), a recessively inherited disease identified by congenital defects, bone marrow failure, infertility, and cancer susceptibility. We confirmed expression and hormonal regulation of Fanca mRNA by quantitative RT-PCR, which showed that GnRH induced a rapid, transient increase in Fanca mRNA. Fanca protein was also acutely upregulated after GnRH treatment of LβT2 cells. In addition, Fanca gene expression was confined to mature pituitary gonadotrophs and adult mouse pituitary and was not expressed in the immature αT3-1 gonadotroph cell line. Thus, this study extends the expression profile of Fanca into a highly specialized endocrine cell and demonstrates hormonal regulation of expression of the Fanca locus. We suggest that this regulatory mechanism may have a crucial role in the GnRH-response mechanism of mature gonadotrophs and perhaps the etiology of FA.

Identification of disease markers by differential display: prion disease.

In order to identify molecular markers of prion disease in peripheral tissues, we used the differential display reverse-transcriptase polymerase chain reaction (DDRT-PCR) procedure to compare gene expression in spleens of infected and uninfected mice. In this study, we identified a novel erythroid-specific gene that was differentially expressed as a result of prion infection. We were able to demonstrate that a decrease in the expression levels of this transcript in hematopoietic tissues was a common feature of prion diseases. Our findings suggest a previously unknown role for the blood erythroid lineage in the development of prion diseases and should provide a new focus for research into diagnostic and therapeutic strategies.