Large non-homology in heteroduplex DNA is processed differently than single base pair mismatches

Summary Unmethylated DNA heteroduplexes with a large single stranded loop in one strand have been prepared from separated strands of DNA from two different strains of bacteriophage , one of which has a 800 base pair IS1 insertion in the cI gene. The results of transfections with these heteroduplexes into wild-type and mismatch repair deficient bacteria indicate that such large non-homologies are not repaired by the Escherichia coli mismatch repair system. However, the results do suggest that some process can act to repair such large non-homologies in heteroduplex DNA. Transfections of a series of recombination and excision repair deficient mutants suggest that known excision or recombination repair systems of E. coli are not responsible for the repair. Repair of large non-homologies may play a role in gene conversion involving large insertion or deletion mutations.     

Coreconstitution of bacterial ATP synthase with monomeric bacteriorhodopsin into liposomes. A comparison between the efficiency of monomeric bacteriorhodopsin and purple membrane patches in coreconstitution experiments

The conditions for coreconstitution of a bacterial ATP synthase and bacteriorhodopsin into lecithin liposomes and for light driven ATP synthesis have been optimized. A rate of maximally 280 nmol ATP min-1 mg ATP synthase-1 was achieved with monomerized bacteriorhodopsin compared with a rate of up to 45 nmol ATP min-1 mg-1 found for proteoliposomes containing bacteriorhodopsin in the form of purple membrane patches. The different rates are explained by the finding that monomeric bacteriorhodopsin is more homogeneously distributed among the liposomes than the purple membrane patches. The final activities depended on both the purification method for the two proteins and the coreconstitution procedure. Furthermore, the ratio (lipid to bacteriorhodopsin to ATP synthase) could be optimized. Light-driven ATP synthesis depends also on the type of detergent used. The best result was obtained by deoxycholate. Also the relationship between proton translocation (by bacteriorhodopsin) and ATP synthesis activity was measured. A constant H+/ATP ratio was found at higher light intensities. This ratio increased strongly at lower light intensities.     

Endothelioma cells expressing the polyoma middle T oncogene induce hemangiomas by host cell recruitment

Mouse endothelioma cells expressing the polyoma middle T oncogene induced hemangiomas in a variety of species such as mice, rats, chicks, and quails. In embryos and newborn mice the hemangiomas expanded within 10-18 hr of injection, disrupting the vasculature and causing the death of the animal. In contrast, the hemangiomas formed a stable structure reminiscent of benign human hemangiomas in adult mice within 5 days. Analysis of the cells comprising the hemangioma revealed that over 95% of the endothelial cells were host derived. No induction of host cell proliferation was detected, and no endothelial mitogens were secreted by the endothelioma cells in vitro. The maintenance of the hemangioma appeared to require the continuous presence of endothelioma cells. The results indicate that these endothelioma cells act as a potent stimulating agent in the rapid formation of hemangiomas by recruiting nonproliferating host endothelial cells.     

Isolation, characterization and immunological determination of basement membrane-associated heparan sulfate proteoglycan

Basement membrane-associated heparan sulfate proteoglycan (HSPG) was extracted from isolated porcine glomerular basement membranes and purified by ion-exchange chromatography. The proteogycan was characterized by specific enzymatic digestions, by amino-acid analysis, by SDS-polyacrylamide gel electrophoresis and by density gradient centrifugation. Polyclonal antibodies were raised against the purified HSPG in rabbits. Antibodies were characterized by enzyme immunoassays, immunoprecipitation and immunohistological methods. They were shown to recognize specifically the core protein of HSPG from porcine, human and rat glomerular basement membrane but did not recognize HSPG from guinea pig or rabbit kidney. The affinity-purified antibodies did not cross-react with other basement membrane proteins like laminin, fibronectin or collagen type IV nor with chondroitin sulfate-rich or keratan sulfate-rich proteoglycans from human or bovine tissue. Using these antibodies an enzyme immunoassay was developed for determination of HSPG in the range of 1-100 ng/ml. Studies with cultured porcine endothelial cells showed that subendothelial basement membrane-associated HSPG may be determined with the enzyme immunoassay.     

Translocation of c-abl oncogene and PDGFB (c-sis) gene in a case of CML with 46,XY, t(22;22)

In a case of CML with a variant Philadelphia translocation (Ph1 or Ph) t(22;22) (q11;q13) in bone marrow cells and unstimulated peripheral blood cells, no cytogenetically detectable involvement of chromosome 9 was observed. Southern blot experiments using probes specific for bcr and c-sis however revealed rearrangement of the bcr, but not of PDGFB (c-sis) gene. Northern blot analysis of bone marrow RNA showed a very weak signal with the c-sis probe, while in a lymph-node biopsy PDGFB m-RNA could not be detected. Chromosomal in situ hybridization gave evidence for translocation of c-abl from chromosome 9 to Ph and of PDGFB from chromosome 22 to chromosome 9, as the result of a threefold translocation t(9;22;22).     

Calcium regulates the rate of rhodopsin disactivation and the primary amplification step in visual transduction

The kinetics of the light-induced activation of transducin as well as the subsequent disactivation process can be monitored by means of a specific light scattering transient PA. In this communication it is demonstrated that the rate of transducin disactivation is calcium dependent, increasing when the calcium concentration is decreased. As a consequence of the accelerated recovery in low calcium, the time to the peak of the transducin activation process is shortened and the gain of the primary amplification step, i.e. the number of transducin molecules activated per bleached rhodopsin, is reduced. Experiments using hydroxylamine as an artificial quencher of rhodopsin activity suggest that calcium acts upon rhodopsin kinase and not upon the rate of the GTPase. This would indicate that calcium may control visual adaptation not only by regulating guanine cyclase activity, but also by affecting the primary step in the transduction cascade, the rhodopsin-transducin coupling.     

Enzyme assay of L-myo-inositol 1-phosphate synthetase based on high-performance liquid chromatography of benzoylated inositol

A procedure for the determination of inositol by reversed-phase HPLC is described which is based on a precolumn benzoylation and detection at 230 nm. This procedure was used to assay the activity of L-myo-inositol 1-phosphate synthetase (EC 5.5.1.4) after treatment of the enzymatic product by a phosphatase.     

Non-C-G recognition sequences of DNA cytosine-5-methyltransferase from rat liver

The eukaryotic DNA cytosine-5-methyltransferase (E.C.2.1.1.37) is known to methylate cytosine in DNA mainly, but not exclusively in C-G. In the present study the minor, non-C-G recognition sequences of a rat DNA methyltransferase were analyzed by Maxam-Gilbert sequencing of in vitro methylated SV40 DNA. The enzyme methylates C-A and C-T at a 50-fold lower initial rate than C-G. Methylation of C-C at the 5'C was not observed in the piece of DNA sequenced. The methylation of C-A is very low in the trinucleotides ACA and CAC, the other C-A containing trinucleotides in DNA are much better methylacceptors. C-T was found methylated predominantly in the sequences CCTAA, ACTAA, and ACTGT. A comparison of the activity with different substrates is in favour of the enzyme making its recognition in the major groove of the DNA.