A rat gene encoding heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

There are at least 3 isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, a bifunctional enzyme which catalyzes the synthesis and degradation of fructose 2,6-bisphosphate. A 22-kb rat gene that encodes the heart isozyme has been identified and compared with the 55-kb rat gene encoding the liver and muscle isozymes which had been described earlier. Although these 2 genes include 12 successive similar exons, they contain dissimilar exons at both ends, consistent with the occurrence of different regulatory domains at the N- and C-termini in the 3 isozymes.     

Cloning and expression in Escherichia coli of a rat hepatoma cell cDNA coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

In liver, the 470-residue bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) catalyses the synthesis and degradation of fructose 2,6-bisphosphate, a potent stimulator of glycolysis. In rat hepatoma (HTC) cells, this enzyme has kinetic, antigenic, and regulatory properties, such as insensitivity to cyclic AMP-dependent protein kinase and lack of associated FBPase-2 activity, that differ from those in liver. To compare the sequence of the HTC enzyme with that of the liver enzyme, we have cloned the corresponding fully-coding cDNA from HTC cells. This cDNA predicts a protein of 448 residues in which the first 32 residues of liver PFK-2/FBPase-2 including the cyclic AMP target sequence have been replaced by a unique N-terminal decapeptide. The rest of the protein is identical with the liver enzyme. An N-terminally truncated recombinant peptide of 380 residues containing the PFK-2 and FBPase-2 domains was expressed in Escherichia coli as a beta-galactosidase fusion protein. It was recognized by anti-PFK-2 antibodies but its enzymic activities were barely detectable. In contrast, a cDNA fully-coding for the HTC enzyme could be expressed in E. coli as a beta-galactosidase-free peptide that exhibited both PFK-2 and FBPase-2 activities. This peptide had those PFK-2 kinetic properties of the HTC enzyme that differ from the liver enzyme. These data, together with immunoblot experiments, suggest that the lack of associated FBPase-2 activity in HTC cells results from a post-translational modification of the enzyme rather than from the difference in amino acid sequence. As well as this peculiar type of PFK-2/FBPase-2 mRNA, HTC cells also contained low concentrations of the liver-type mRNA. Unlike in liver, neither mRNA was induced by dexamethasone in these cells.     

New informative polymorphism at the DXS304 locus,a close distal marker for the fragile X locus

Summary The polymorphic DNA marker DXS304 detected by probe U6.2 has recently been shown to be closer to the fragile X locus than previously available markers. Its usefulness has however been limited by its relatively low heterozygosity. We have isolated, by cosmid cloning, a 67 kilobase region around probe U6.2 and have characterized a new probe (U6.2-20E) that detects BanI and BstEII restriction fragment length polymorphisms (RFLPs). The BanI RFLP has a heterozygosity of 0.49 and is in partial linkage disequilibrium with the previously described polymorphism, with a combined heterozygosity of 0.63. Furthermore, we have found that the U6.2 original probe, which probably detects an insertion-deletion polymorphism, is also informative in BanI digests. Thus, the two informative RFLPs at the DXS304 locus can be conveniently tested in a single hybridization with a single digest. An updated linkage analysis confirms that DXS304 is distal to the fragile X locus. This informative locus can now be used effectively for genetic mapping of the Xq27–q28 region, and for diagnostic applications in fragile X or Hunter syndrome families.     

Chromosome localization and polymorphism of an oestrogen-inducible gene specifically expressed in some breast cancers

Summary The BCEI gene codes for a small secreted protein and is expressed in the human mammary tumour cell line MCF7 under oestrogen control and in some breast cancers. We have mapped the gene to chromosome 21 using a panel of somatic hybrid lines, and in situ hybridization has allowed a precise assignment to band 21q223. Two restriction fragment length polymorphisms (RFLP) are described that should be of use in linkage or population studies to test a possible involvement of the BCEI gene in genetic predisposition to breast cancer. This gene should also be a useful marker for the genetic and physical mapping of chromosome 21, and for a better definition of the region involved in the clinical phenotype of Downs syndrome.     

A catalogue of Clostridium thermocellum endoglucanase, β-glucosidase and xylanase genes cloned in Escherichia coli

Abstract Two independent collections of clones containing Clostridium thermocellum genes involved in cellulose have been previously obtained at IAPGR, Cambridge, and at the Pasteur Institute, Paris. The two collections were compared for cross-hybridization, restriction maps and enzyme phenotypes. Truly distinct genes were one β-glucosidase gene, two xylanase genes, and fifteen endogluconase genes. Two of the cloned fragments contained extraneous DNA which was absent from their respective counterparts isolated in the other collection. The dicrepancies resulted from in vivo rearrangements which had occurred in either of the C. thermocellum NCIB 10682 stocks used to generate the two gene banks.     

Monoamine Oxidase-Inhibiting Properties of SR 95191, a New Pyridazine Derivative, in the Rat: Evidence for Selective and Reversible Inhibition of Monoamine Oxidase Type A In Vivo but Not In Vitro

In rodents, SR 95191 [3-(2-morpholinoethylamino)-4-cyano-6-phenylpyridazine] has been shown to be active in animal models of depression. The profile of activity of SR 95191 suggests that the compound is a selective and short-acting type A monoamine oxidase (MAO) inhibitor (MAOI) in vivo. In the present study, the interaction of SR 95191 with MAO-A and MAO-B activity was further examined in vivo and in vitro. In brain, liver, and duodenum of pretreated rats, SR 95191 selectively inhibited MAO-A (ED50 = 3-5 mg/kg, p.o.), whereas MAO-B was only weakly inhibited for doses as high as 300 mg/kg, p.o. In vivo, SR 95191 (1-100 mg/kg, p.o.) antagonized, in a dose-dependent fashion, the irreversible inhibition of brain and liver MAO-A induced by phenelzine. Finally, dopamine and 5-hydroxytryptamine depleted from their striatal stores by tetrabenazine were able to displace SR 95191 from the active site of MAO-A. However, ex vivo, kinetic studies showed that the inhibitory effect of SR 95191 (1-10 mg/kg) towards MAO-A was noncompetitive and was unchanged after dilution or dialysis. In vitro, the inhibition of brain MAO-A, but not MAO-B, by SR 95191 was time dependent, with a 19-fold decrease in the IC50 values being observed over a 30-min incubation period (140 to 7.5 microM). At this time, the SR 95191-induced inhibition of MAO-A was not removed by repeated washings. When the reaction was started by adding the homogenate without prior preincubation with SR 95191, the inhibition of brain MAO-A was fully competitive (Ki = 68 microM).(ABSTRACT TRUNCATED AT 250 WORDS)