Biomedical subjects
Publications and source records attributed to P Manlapaz-Ramos.
We have presented restriction analyses which indicate that the first transposition event after induction can be carried out and analyzed in vitro using cellophane disc lysates. This would be a first step in describing in molecular terms a "wiring diagram" of replicative transposition.
An in vitro system for bacteriophage Mu DNA replication using lysates on cellophane discs is described. Mu replication was monitored by DNA hybridization. Using a thermoinducible Mu lysogen, 30-50% of all DNA synthesis in vitro was Mu-specific. Mu DNA synthesis is semidiscontinuous. In the presence of the DNA ligase inhibitor NMN, about one-half of the DNA was in Okazaki pieces and one-half in large DNA. The Mu Okazaki pieces hybridized mainly to the Mu light strand; the large DNA hybridized mainly to the Mu heavy strand. Okazaki pieces isolated from uninfected cells also hybridized to 2000-3000 bases of host DNA present in Mu-separated strands. However, the host Okazaki pieces hybridize to both Mu strands symmetrically. Most, if not all, host sequences were represented in mature Mu viral DNA. The in vitro data are most consistent with models in which Mu sequences, oriented randomly in both directions in the host chromosome, have recruited a bacterial replisome which traverses the Mu genome from left to right.
Mu DNA replication has been carried out in vitro on cellophane discs in the presence of dBUTP. If the DNA is sheared to 80 kb pieces, the Mu replicas band anomalously in CsCl gradients between hybrid and light DNA density positions. The intermediate density DNA comprises semiconservatively replicated Mu sequences, flanked by unreplicated light DNA. This and previous data are consistent with replication occurring within Mu boundaries. Both the synthesis of Mu sequences and the intermediate density DNA are abolished by protein synthesis inhibition in vivo just prior to lysis on cellophane discs. These observations indicate that at least some steps in bona fide Mu transposition-replication are being observed in vitro.
A previously undescribed nucleoside salvage pathway for NAD biosynthesis is defined in Salmonella typhimurium. Since neither nicotinamide nor nicotinic acid is an intermediate in this pathway, this second pyridine nucleotide salvage pathway is distinct from the classical Preiss-Handler pathway. The evidence indicates that the pathway is from nicotinamide ribonucleoside to nicotinamide mononucleotide (NMN) and then to nicotinic acid mononucleotide, followed by nicotinic acid adenine dinucleotide and NAD. The utilization of exogenous NMN for NAD biosynthesis has been reexamined, and in vivo evidence is provided that the intact NMN molecule traverses the membrane.
Different metabolic steps comprise the pyridine nucleotide cycles in Escherichia coli and in the human cell line HeLa D98/AH2. An analysis of the 32P-labeling patterns in vivo reveals that in E. coli, pyrophosphate bond cleavage of intracellular NAD predominates, while in the human cell line, cleavage of the nicotinamide ribose bond predominates. In E. coli, intracellular NAD is processed differently from extracellular NAD. Conversion of intracellular NAD to nicotinic acid mononucleotide (NaMN) can be demonstrated in intact cells. We have also assayed and purified an enzyme, NMN deamidase, which converts NMN to NaMN. These data suggest that in E. coli, the predominant intracellular pyridine nucleotide cycle operative under our experimental conditions is: NAD leads to NMN leads to NaMN leads to NaAD leads to NAD Thus, a metabolic event requiring pyrophosphate bond cleavage of NAD, such as DNA ligation, initiates most NAD turnover. In the human cell line, the data are consistent with the following NAD turnover cycle: (formula, see text) Whereas in E. coli, ADP-ribosylation does not make a quantitatively important contribution, we suggest that in HeLa cells, ADP-ribosylation events initiate NAD turnover.
Explore the source record for details and available documents.
Explore the source record for details and available documents.