A striking example of the relationship between regulation of transcription and phenotype is the central role of the Y-chromosomal gene Sry in mammalian sex determination. Sry is the founding member of a large family of so-called Sox genes. During murine embryogenesis, the transcriptional activator Sox-4 is expressed at several sites, but in adult mice expression is restricted to immature B and T lymphocytes. Using targeted gene distruption, we have found that SOX-4(-/-) embryos succumb to circulatory failure at day E14. This was a result of impaired development of the endocardial ridges (a specific site of Sox-4 expression) into the semilunar valves and the outlet portion of the muscular ventricular septum. The observed range of septation defects is known as 'common arterial trunk' in man. We studied haemopoiesis in lethally irradiated mice reconstituted with SOX-4(-/-) fetal liver cells and found that a specific block occurred in B-cell development at the pro-B cell stage. In line with this, the frequency and proliferative capacity of IL-7-responsive B cell progenitors in fetal liver were severely decreased in vitro.

Cell division in Escherichia coli requires the products of the ftsQ, ftsA and ftsZ genes. It is not known how the cell regulates the cellular concentrations of these essential elements of the division system. We describe here a factor that activates cell division by specifically increasing transcription from one of the two promoters that lie immediately upstream of the ftsQAZ gene cluster. The trans‐acting factor is the product of the sdiA gene, which was isolated on the basis of its ability to suppress the division inhibitory effect of the MinC/MinD division inhibitor. In addition, the sdiA gene product suppressed the action of other chromosomally encoded division inhibitors, induced minicell formation in wild type cells, and restored division activity to an ftsZ temperature‐sensitive mutant grown under nonpermissive conditions. All of these properties were explained by the ability of the sdiA gene product specifically to increase transcription of the ftsQAZ gene cluster, resulting in an increase in cellular concentration of the FtsZ protein. The sdiA gene product is the first factor thus far identified that specifically regulates expression of this key group of cell division genes.

During development fast-contracting atrial and ventricular chambers develop from a peristaltic-contracting heart tube. This study addresses the question of whether chamber formation is paralleled by a matching expression of the sarcoplasmic reticulum (SR) Ca(2+) pump. We studied indo-1 Ca(2+) transients elicited by field stimulation of linear heart tube stages and of explants from atria and outflow tracts of the prototypical preseptational E13 rat heart. Ca(2+) transients of H/H 11+ chicken hearts, which constitute the prototypic linear heart tube stage, were sensitive to verapamil only, indicating a minor contribution of Ca(2+)-triggered SR Ca(2+) release. Outflow tract transients displayed sensitivity to the inhibitors similar to that of the linear heart tube stages. Atrial Ca(2+) transients disappeared upon addition of ryanodine, tetracaine, or verapamil, indicating the presence of Ca(2+)-triggered SR Ca(2+) release. Quantitative radioactive in situ hybridization on sections of E13 rat hearts showed approximately 10-fold higher SERCA2a mRNA levels in the atria compared to nonmyocardial tissue and approximately 5-fold higher expression in compact ventricular myocardium. The myocardium of atrioventricular canal, outflow tract, inner curvature, and ventricular trabecules displayed weak expression. Immunohistochemistry on sections of rat and human embryos showed a similar pattern. The significance of these findings is threefold. (i) A functional SR is present long before birth. (ii) SR development is concomitant with cardiac chamber development, explaining regional differences in cardiac function. (iii) The pattern of SERCA2a expression underscores a manner of chamber development by differentiation at the outer curvature, rather than by segmentation of the linear heart tube.

We studied the distribution of the mRNAs for carbamoylphosphate synthetase (ammonia) and glutamine synthetase in frozen sections of adult rat liver by in situ hybridization to [35S]-labeled cDNA probes. The density of silver grains resulting from hybridization to the labeled cDNA probe for carbamoylphosphate synthetase is highest around the portal venules, decreases towards the central venule, and is virtually absent from an area two to three cells wide that lines the central venules in which mRNA for glutamine synthetase is predominantly localized. Therefore, both mRNAs show the same complementary distribution within the liver acinus that was found for the proteins they encode, demonstrating that compartmentalization of the expression of these enzymes is controlled at a pretranslational level. In addition, we found that carbamoylphosphate synthetase mRNA is present mainly in the epithelium of the crypts of the proximal part of the small intestine, whereas carbamoylphosphate synthetase protein is present in the epithelium of both crypts and villi.

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The carbamoyl-phosphate synthetase I gene is expressed in the periportal region of the liver, where it is activated by glucocorticosteroids and glucagon (via cyclic AMP), and in the crypts of the intestinal mucosa. The enhancer of the gene is located 6.3 kilobase pairs upstream of the transcription start site and has been shown to direct the hormone-dependent hepatocytespecific expression in vitro. To analyze the function of the upstream region in vivo, three groups of transgenic mice were generated. In the first group the promoter drives expression of the reporter gene, whereas the promoter and upstream region including the far upstream enhancer drive expression of the reporter gene in the second group. In the third group the far upstream enhancer was directly coupled to a minimized promoter fragment. Reporter-gene expression was virtually undetectable in the first group. In the second group spatial, temporal, and hormonal regulation of expression of the reporter gene and the endogenous carbamoyl-phosphate synthetase gene were identical. The third group showed liver-specific periportal reporter gene expression, but failed to activate expression in the intestine. These results show that the upstream region of the carbamoyl-phosphate synthetase gene controls four characteristics of its expression: tissue specificity, spatial pattern of expression within the liver and intestine, hormone sensitivity, and developmental regulation. Within the upstream region, the far upstream enhancer at ؊6.3 kilobase pairs is the determinant of the characteristic hepatocyte-specific periportal expression pattern of carbamoyl-phosphate synthetase.The ornithine cycle converts ammonia, originating mainly from amino acid metabolism, into urea for excretion. This conversion is vital, because ammonia is toxic to vertebrates. All five ornithine cycle enzymes are active predominantly in the periportal hepatocytes (1, 2, 68). Parts of the cycle, however, are also found in the intestinal mucosa and the kidney, thereby creating an interorgan biosynthetic pathway for arginine biosynthesis (reviewed in Refs. 1, 3, and 4). The first and, under most conditions, major flux-determining step of the ornithine cycle, the conversion of ammonia, bicarbonate, and ATP into carbamoyl phosphate, is catalyzed by carbamoyl-phosphate synthetase I (CPS; EC 6.3.4.16) 1 (1, 5). The expression of CPS mRNA can be detected in the liver of the rat from embryonic day 15 (ED15) onwards. Expression is initially found only in a few hepatocytes, but toward the end of the fetal period all hepatocytes have been recruited to express CPS (6 -8). Subsequently, the expression gradually becomes confined to the hepatocytes surrounding the portal veins (8 -10). CPS enzyme and mRNA levels in liver change in parallel under experimental conditions that change circulating glucocorticosteroid and hepatic cyclic AMP levels (3,(11)(12)(13)(14), suggesting that the zonal restriction and hormonal modulation of CPS expression are regulated at the level of transcription. Accordingly, it was s...

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Densitometry on autoradiographs of sections processed for in situ hybridization provides a direct measure for the in situ quantification of mRNA. Gelatin spots, containing different concentrations of the radioisotope, and processed in parallel with the tissue sections, can be used as a sensitive model to calibrate the densitometric measurements. The shape of the gelatin spots was shown to be circular with a parabolic crosssectional profile. This simple shape allows the subdivision of the spot into a series of concentric rings, which enables an unbiased measurement of the optical density -radioactivity relation. This spot measurement is also applicable to DNA arrays spotted on glass or membranes. A new model, explaining the optical density of autoradiographs, was derived and fitted to the calibration points. The use of this calibration method is crucial for the correct interpretation of autoradiographs