Identifying essential genes in bacterial metabolic networks with machine learning methods

Background  

Identifying essential genes in bacteria supports to identify potential drug targets and an understanding of minimal requirements for a synthetic cell. However, experimentally assaying the essentiality of their coding genes is resource intensive and not feasible for all bacterial organisms, in particular if they are infective.     

Pleiotropic mutations in Serratia marcescens which increase the synthesis of certain exocellular proteins and the rate of spontaneous prophage induction

Summary Mutants of S. marcescens HY have been isolated which produce between five and one hundred times more exocellular nuclease than does the parental strain. These nuclease-superactive (nuc ^su) mutants are highly pleiotropic: they produce more exocellular marcescin A and lipase than the wild-type and their ability to inactivate penicillin G is increased. Furthermore, all nuclease-superactive mutants if lysogenic for the heteroimmune phages Kappa and/or y show spontaneous induction rates for both prophages 10 to 200 fold greater than the corresponding wild-type. Nuc ^su mutants of independent origin synthesize nuclease and marcescin A in approximately proportional amounts although the corresponding structural genes do not seem to be part of a single operon because some bacteriocin-superactive mutants were isolated which showed an increase of the synthesis of marcescin A only. Nuclease-defective (nuc) mutants are all of the non-pleiotropic type. Three hypotheses to explain the effects of the nuc ^su mutation at the molecular level are discussed and some evidence in support of one of these hypotheses (gene-dosage effect) is presented in an accompanying paper (Timmis and Winkler, 1973).     

Exponential Megapriming PCR (EMP) Cloning—Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints

We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.     

Determination of deuterium-labeled tryptophan in proteins by means of high-performance liquid chromatography and thermospray mass spectrometry

In order to develop direct methods for determining the extent of metabolic incorporation of isotopically labeled amino acids into a protein, the determination of deuterated tryptophan in [2H5]tryptophan-bacteriorhodopsin was investigated. The isotopically modified protein was subjected to alkaline hydrolysis. After phenyl isothiocyanate derivatization of the hydrolysate, the mixture was separated by reversed-phase liquid chromatography. Field desorption mass spectrometry and thermospray mass spectrometry were investigated for their ability to determine the ratio between [2H5]tryptophan and total tryptophan in the collected fractions. In order to check the procedure a set of known tryptophan/[2H5]tryptophan mixtures were passed through the same derivatization, HPLC separation, and lyophilization procedure as used for the biological samples.     

Fibronectin initiates fibrin production by rabbit corneal endothelial cells in vitro

Confluent rabbit corneal endothelial cells incubated in the absence of serum do not produce fibrinogen. When exogenous fibronectin is added to these cultures, fibrinogen production is observed. Fibronectin concentrations stimulate fibrinogen synthesis by endothelial cells in a dose-response fashion. This direct interaction of fibronectin and fibrinogen may be important in both wound healing processes and pathological states.