Genetic engineering of peptide hormones

The application of different approaches for preparing DNAs coding for peptide hormones was demonstrated. The libraries of human, bovine and porcine pituitaries cDNA were obtained starting from their total mRNAs. Screening of these libraries revealed clones containing human, bovine and porcine growth hormone sequences, cDNAs for bovine ACTH-beta-lipotropin precursor and for bovine and porcine prolactin. The gene of human calcitonin was created by combination of chemical and enzymatic synthesis. This synthetic gene was further cloned in pBR322. The expression of cloned human growth hormone cDNA under control of different Escherichia coli promoters was studied and physico-chemical and biological properties of the growth hormone produced by E. coli were tested.     

Developmental abnormalities in mice transgenic for bovine oncostatin M.

Oncostatin M belongs to the subfamily of hematopoietin cytokines that binds a receptor complex containing gp130. To date, only the human form of oncostatin M has been identified, and its evolutionary conservation is unresolved. We have isolated a bovine gene whose open reading frame encodes a precursor protein that is 58% identical to human oncostatin M. A comparison of the bovine and human amino acid sequences predicts significant similarity, including the four-alpha-helical-bundle structure and the placement of disulfide bridges. As with the human protein, bovine oncostatin M binds specific receptors on human H2981 cells and inhibits the proliferation of human A375 tumor cells and mouse M1 leukemia cells. To identify activities regulated in vivo, we injected bovine oncostatin M fusion genes containing various tissue-specific promoters into mouse embryos. The frequencies of transgenic mice were reduced significantly, suggesting that overexpression of the bovine cytokine is detrimental to normal mouse development. In addition to deaths associated with expression in neurons and keratinized epithelia, bovine oncostatin M caused abnormalities in bone growth and spermatogenesis, stimulated fibrosis surrounding islets in the pancreas, and disrupted normal lymphoid tissue development. This work establishes the existence of a nonprimate oncostatin M gene and provides the first demonstration that this cytokine can function in a pleiotropic manner in vivo. Information regarding bovine oncostatin M may help characterize the structure and function of this cytokine in other vertebrate species.     

Hormonal Regulation of Opioid Peptide Neurons in the Anteroventral Periventricular Nucleus

Steroid hormones provide a means of coordinating the activity of widespread neural systems that mediate endocrine, autonomic, and somatomotor aspects of reproductive processes that are essential for the propagation of mammalian species. Because these processes are quite different in each sex, the neural pathways that control them are also sexually differentiated. The anteroventral periventricular nucleus (AVPV) of the preoptic region occupies a nodal point in sexually dimorphic forebrain circuits and appears to play a critical role in regulating gonadotropin secretion. The AVPV contains sexually dimorphic populations of opioid peptide containing neurons that display different patterns of development and are differentially regulated in adult animals by gonadal steroids. Moreover, estrogen (ER) and progesterone (PR) receptors are expressed in AVPV neurons in a transmitter-specific way, and the expression of these nuclear transacting factors is differentially regulated by sex steroids. Thus, neurons in the AVPV show distinct patterns of hormonal regulation of gene expression, and distinct hormone receptor profiles.     

Differentiation of murine embryonic stem cells induces progesterone receptor gene expression

The role of steroid hormone receptors in very early embryonic development remains unknown. Clearly, expression during organogenesis is important for tissue-specific development. However, progesterone receptor (PR) and estrogen receptors (ERalpha, ERbeta) are expressed during early development through the blastocyst stage in mice and other species, and yet are not essential for embryonic viability. We have utilized the mouse embryonic stem (mES) cell model to investigate the regulated expression of these receptors during differentiation. Surprisingly, one of the earliest changes in gene expression in response to a differentiation signal observed is PR gene induction. It parallels the time course of expression for the patterning genes Hoxb1 and Hoxa5. Unexpectedly, PR gene expression is not regulated in an estrogen-dependent manner by endogenous ERs or by transiently overexpressed ERalpha. Our results suggest a potentially novel mechanism of PR gene regulation within mES cells compared to adult tissues and the possibility of unique targets of PR action during early mES cell differentiation.     

Regulation of expression of the genes encoding steroidogenic enzymes

In recent years it has become apparent that tropic hormones involved in steroidogenesis act to regulate the expression of the enzymes involved in the various steroidogenic pathways. This is particularly evident in the ovary where the episodic secretion of steroids throughout the ovarian cycle is regulated largely by changes in the levels of the particular enzymes involved in each step of the steroid biosynthetic pathways. Recently, the genes for the various cytochrome P450 species involved in ovarian steroidogenesis, namely cholesterol side-chain cleavage P450 (P450SCC), 17 alpha-hydroxylase P450 (P450(17 alpha], and aromatase cytochrome P450 (P450AROM) have been isolated and characterized, making it possible to study the regulation of expression at the molecular level. To this end, a series of chimeric constructs have been prepared in which fragments of the 5'-untranslated region of bovine P450(17 alpha) and P450SCC have been inserted upstream of the chloramphenicol acetyl transferase (CAT) and beta-globin reporter genes. These constructs have been used to transfect primary cultures of bovine luteal and thecal cells. The results indicate that cAMP responsiveness lies within defined regions of genes which do not contain a classical CRE, similar to previous results utilizing adrenal cells in culture. Furthermore, although constructs containing both the P450(17 alpha) and P450SCC 5'-upstream regions are expressed in both luteal and thecal cell cultures, only those containing the P450SCC sequences are expressed in luteal cells. Studies on the expression of P450AROM indicate that the promoter which is responsible for its expression in human placenta is not operative in the corpus luteum. Thus estrogen biosynthesis may be regulated by the differential use of tissue specific promoters, thus accounting for the complexity and multifactorial nature of the expression of this activity.     

Thyroid hormone regulation of hepatic genes in vivo detected by complementary DNA microarray

The liver is an important target organ of thyroid hormone. However, only a limited number of hepatic target genes have been identified, and little is known about the pattern of their regulation by thyroid hormone. We used a quantitative fluorescent cDNA microarray to identify novel hepatic genes regulated by thyroid hormone. Fluorescent-labeled cDNA prepared from hepatic RNA of T3-treated and hypothyroid mice was hybridized to a cDNA microarray, representing 2225 different mouse genes, followed by computer analysis to compare relative changes in gene expression. Fifty five genes, 45 not previously known to be thyroid hormone-responsive genes, were found to be regulated by thyroid hormone. Among them, 14 were positively regulated by thyroid hormone, and unexpectedly, 41 were negatively regulated. The expression of 8 of these genes was confirmed by Northern blot analyses. Thyroid hormone affected gene expression for a diverse range of cellular pathways and functions, including gluconeogenesis, lipogenesis, insulin signaling, adenylate cyclase signaling, cell proliferation, and apoptosis. This is the first application of the microarray technique to study hormonal regulation of gene expression in vivo and should prove to be a powerful tool for future studies of hormone and drug action.