Accumulation of carbamoylphosphate-synthetase and phosphoenolpyruvate-carboxykinase mRNA in embryonic rat hepatocytes. Evidence for translational control during the initial phases of hepatocyte-specific gene expression in vitro

The aim of this study was to establish whether the initial accumulation of hepatocyte-specific proteins after hormone induction is regulated at the pretranslational and/or the translational level. To this end, mRNA molar concentrations were determined and compared with rates of protein synthesis from previous studies [van Roon, M.A., Charles, R. & Lamers, W.H. (1987) Eur. J. Biochem. 165, 229-234]. In vivo, carbamoylphosphate-synthetase mRNA starts to accumulate at day 17 of pregnancy. Phosphoenolpyruvate-carboxykinase mRNA starts to accumulate only just prior to birth. Embryonic day 14 (i.e. 8 days before the expected day of birth), livers were chosen to study the regulation of the initiation of hepatocyte-specific mRNA accumulation in vitro. Accumulation of carbamoylphosphate-synthetase and phosphoenolpyruvate-carboxykinase mRNA is regulated by the same hormones as accumulation of the respective proteins. The rate at which carbamoylphosphate-synthetase and phosphoenolpyruvate-carboxykinase mRNA molecules accumulate in cultured embryonic hepatocytes is relatively low, compared to that of postnatal hepatocytes. However, the increase of the rate of synthesis of carbamoylphosphate-synthetase and phosphoenolpyruvate-carboxykinase protein is even 3-6-fold slower than that of mRNA. This shows that initially mRNAs accumulate intracellularly to a relatively high concentration without being efficiently translated or translatable. Only after the mRNA concentration reaches a plateau of 72 h and 48 h respectively, the cellular capacity to synthesize the respective proteins increases. Therefore, the translational efficiency is certainly one of the major rate-limiting factors of the initial phases of expression of the hepatocyte-specific genes for carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase.     

ATP increases the affinity between MutS ATPase domains. Implications for ATP hydrolysis and conformational changes

MutS is the key protein of the Escherichia coli DNA mismatch repair system. It recognizes mispaired and unpaired bases and has intrinsic ATPase activity. ATP binding after mismatch recognition by MutS serves as a switch that enables MutL binding and the subsequent initiation of mismatch repair. However, the mechanism of this switch is poorly understood. We have investigated the effects of ATP binding on the MutS structure. Crystallographic studies of ATP-soaked crystals of MutS show a trapped intermediate, with ATP in the nucleotide-binding site. Local rearrangements of several residues around the nucleotide-binding site suggest a movement of the two ATPase domains of the MutS dimer toward each other. Analytical ultracentrifugation experiments confirm such a rearrangement, showing increased affinity between the ATPase domains upon ATP binding and decreased affinity in the presence of ADP. Mutations of specific residues in the nucleotide-binding domain reduce the dimer affinity of the ATPase domains. In addition, ATP-induced release of DNA is strongly reduced in these mutants, suggesting that the two activities are coupled. Hence, it seems plausible that modulation of the affinity between ATPase domains is the driving force for conformational changes in the MutS dimer. These changes are driven by distinct amino acids in the nucleotide-binding site and form the basis for long-range interactions between the ATPase domains and DNA-binding domains and subsequent binding of MutL and initiation of mismatch repair.     

Vertical Signalling Involves Transmission of Hox Information from Gastrula Mesoderm to Neurectoderm

Development and patterning of neural tissue in the vertebrate embryo involves a set of molecules and processes whose relationships are not fully understood. Classical embryology revealed a remarkable phenomenon known as vertical signalling, a gastrulation stage mechanism that copies anterior-posterior positional information from mesoderm to prospective neural tissue. Vertical signalling mediates unambiguous copying of complex information from one tissue layer to another. In this study, we report an investigation of this process in recombinates of mesoderm and ectoderm from gastrulae of Xenopus laevis. Our results show that copying of positional information involves non cell autonomous autoregulation of particular Hox genes whose expression is copied from mesoderm to neurectoderm in the gastrula. Furthermore, this information sharing mechanism involves unconventional translocation of the homeoproteins themselves. This conserved primitive mechanism has been known for three decades but has only recently been put into any developmental context. It provides a simple, robust way to pattern the neurectoderm using the Hox pattern already present in the mesoderm during gastrulation. We suggest that this mechanism was selected during evolution to enable unambiguous copying of rather complex information from cell to cell and that it is a key part of the original ancestral mechanism mediating axial patterning by the highly conserved Hox genes.     

Unique and shared functions of different connexins in mice

BACKGROUND: Connexins are the protein subunits of intercellular gap junction channels. In mammals, they are encoded by a family of at least 15 genes, which show cell-type-specific but overlapping patterns of expression. Mice lacking connexin43 (Cx43) die postnatally from obstruction of the right ventricular outflow tract of the heart. To discriminate between the unique and shared functions of Cx43, Cx40 and Cx32, we generated two 'knock-in' mouse lines, Cx43KI32 and Cx43KI40, in which the coding region of the Cx43 gene was replaced, respectively, by the coding regions of Cx32 or Cx40. RESULTS: Heterozygous mutants were fertile and co-expressed the wild-type and the corresponding recombinant allele in all tissues analyzed. Heterozygous Cx43KI32, but not Cx43KI40, mutant mothers were unable to nourish their pups to weaning age, possibly reflecting a defect in milk ejection. Homozygous mutant males were sterile because of extensive germ-cell deficiency. The ovaries of homozygous Cx43KI32 neonates exhibited all stages of follicular development and ovulation. The hearts of homozygous Cx43KI32 neonates showed mild morphological defects, but the cardiac morphology of homozygous Cx43KI40 neonates was relatively normal. Spontaneous ventricular arrhythmias were observed in most Cx43KI40 and some Cx43KI32 mutant mice, suggesting increased ventricular vulnerability in these mice. CONCLUSIONS: The postnatal lethality of Cx43-deficient mice was rescued in Cx43KI32 or Cx43KI40 mice, indicating that Cx43, Cx40 and Cx32 share at least some vital functions. On the other hand, Cx43KI32 and Cx43KI40 mice differed functionally and morphologically from each other and from wild-type mice. Thus, these connexins also have unique functions.     

Architecture of the Pol III–clamp–exonuclease complex reveals key roles of the exonuclease subunit in processive DNA synthesis and repair

DNA polymerase III (Pol III) is the catalytic α subunit of the bacterial DNA Polymerase III holoenzyme. To reach maximum activity, Pol III binds to the DNA sliding clamp β and the exonuclease ε that provide processivity and proofreading, respectively. Here, we characterize the architecture of the Pol III–clamp–exonuclease complex by chemical crosslinking combined with mass spectrometry and biochemical methods, providing the first structural view of the trimeric complex. Our analysis reveals that the exonuclease is sandwiched between the polymerase and clamp and enhances the binding between the two proteins by providing a second, indirect, interaction between the polymerase and clamp. In addition, we show that the exonuclease binds the clamp via the canonical binding pocket and thus prevents binding of the translesion DNA polymerase IV to the clamp, providing a novel insight into the mechanism by which the replication machinery can switch between replication, proofreading, and translesion synthesis.     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Developmental and hormonal regulation of carbamoyl-phosphate synthase gene expression in rat liver: evidence for control mechanisms at different levels in the perinatal period

Carbamoyl-phosphate synthase gene expression is found to be primarily regulated by conditions that enhance hepatic glucocorticosteroid levels (hormone injections) and cyclic AMP levels (induction of diabetes). After birth, changes in the level of carbamoyl-phosphate synthase protein follow changes in the level of carbamoylphosphate synthase mRNA, suggesting a pretranslational control mechanism. In fetal rats, carbamoyl-phosphate synthase gene expression is regulated by the same factors as in adults. However, both the level to which carbamoyl-phosphate synthase mRNA can accumulate and the extent to which mRNA can be translated appear to be limited, indicating control mechanisms at the pretranslational and translational level. Finally, in the immediate postnatal period, a transient but pronounced decrease in the rate of degradation of carbamoyl-phosphate synthase protein may play a role in the accumulation of the enzyme.