An expression cassette carrying 426 basepairs of the rat probasin (PB) gene promoter and 28 basepairs of 5'-untranslated region is sufficient to target the expression of the bacterial chloramphenicol acetyltransferase (CAT) gene specifically to the prostate in transgenic mice. The PB-CAT transgene was expressed in three of five (60%) independent lines of mice, and this expression, as reported previously for the endogenous rat gene, was male specific, restricted primarily to the lateral, dorsal, and ventral lobes of the prostate, with only very low levels of CAT activity detected in the anterior prostate and seminal vesicles. The developmental and hormonal regulation of the transgene also paralleled that reported for the rat gene, with a 70-fold increase in CAT activity in the mouse prostate observed between 2-7 weeks of age, a time corresponding to sexual maturation. PB-CAT activity in the prostate declined after castration to 3.5% of the precastration level, and the CAT activity in castrated males approached precastration levels when mice were supplemented with testosterone. Transgene expression in castrated males was not induced by dexamethasone. Coinjection of PB-CAT with a chicken lysozyme gene matrix attachment region resulted in their cointegration and further restricted the pattern of PB-CAT to the dorsolateral prostate, with suppressed expression observed in the ventral prostate. These studies demonstrate that a minimal rat probasin promoter can target heterologous gene expression specifically to the prostate in a developmentally and hormonally regulated fashion.

A complementary DNA (cDNA) encoding rat insulin-like growth factor 1 (IGF-I) has been isolated from a kidney cDNA library. By analogy with the sequence of human and mouse preproIGF-IA this cDNA encodes rat preproIGF-I from amino acid minus 3 to amino acid 105. The predicted protein sequence shows 96% and 99% homology with the human and mouse preproIGF-I, respectively. Under stringent conditions the rat IGF-I cDNA hybridizes with at least three mRNA, messenger RNA species which have apparent sizes of 7, 1.8, and 0.7-1.1 kilobases. All three IGF-I transcripts are detectable in each of the normal rat tissues examined and the relative order of abundance in tissues from intact adult male rats is liver greater than lung greater than kidney greater than thymus greater than spleen greater than heart greater than skeletal muscle (quadriceps femoris) greater than testes greater than brain. The GH dependence of the IGF-I mRNAs was demonstrated by the administration of a single ip injection of human GH (hGH) to hypophysectomized (hypox) rats which resulted in an increase in all three transcripts in each of the tissues examined. The increase in IGF-1 mRNAs was most marked in skeletal and cardiac muscle (9.7- and 9.5-fold compared to hypox controls, respectively) and least marked in the brain. In the liver only a 4-fold increase in IGF-I expression was observed, possibly because of the relatively high level of IGF-I expression in the tissue in the hypox control rats. Each of the IGF-I messenger RNAs appeared to increase in parallel and the time course of IGF-I induction was similar in each tissue with maximal levels of IGF-I transcripts present 6 to 12 h after GH administration. A dose-dependent increase in IGF-I mRNAs was observed in most tissues of hypox rats treated for 10 days with hGH. Significant correlations between growth, as determined by body weight gain and IGF-I expression were observed in liver (r = 0.97), kidney (r = 0.90), quadriceps femoris (r = 0.95), diaphragm (r = 0.92), and thymus (r = 1.0). The IGF-I mRNA levels in tissues from rats that had been treated with the highest dose of hGH (90 microgram/rat X day) were similar to those observed in normal intact rats. This study confirms the highly conserved nature of the IGF-I precursor and provides clear evidence for the GH dependence of IGF-I gene expression in multiple tissues of the rat.

Fibroblast growth factor 16 (FGF-16) expression has previously been detected in mouse heart at mid-gestation in the endocardium and epicardium, suggesting a role in embryonic heart development. More specifically, exogenously applied FGF-16 has been shown to stimulate growth of embryonic myocardial cells in tissue explants. We have generated mice lacking FGF-16 by targeting the Fgf16 locus on the X chromosome. Elimination of Fgf16 expression resulted in embryonic death as early as day 11.5 (E11.5). External abnormalities, including hemorrhage in the heart and ventral body region as well as facial defects, began to appear in null embryos from E11.5. Morphological analysis of FGF-16 null hearts revealed cardiac defects including chamber dilation, thinning of the atrial and ventricular walls, and poor trabeculation, which were visible at E10.5 and more pronounced at E11.5. These findings indicate FGF-16 is required for embryonic heart development in mid-gestation through its positive effect on myocardial growth.

Efficient mechanised synthesis of heptadecadeoxyribonucleotides has been achieved on an economically small scale by an improved solid phase phosphotriester method on a polydimethylacrylamide resin. Improvements were made in the preparation of dinucleotide building blocks, reaction conditions for oligonucleotide assembly and in purification of deprotected oligonucleotides by h.p.l.c. Several milligrams of pure heptadecamers were obtained. Two of the heptadecamers were designed for sequencing in opposite directions of DNA cloned in phage M13mp2.

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