Belonging and community: understandings of "home" and "friends" among the English poor, 1750-1850.

This article is based on unique 'narratives of the poor', that is, letters from poor people to their parishes of settlement, petitions to the London Refuge of the Destitute, and letters from mothers to the London Foundling Hospital, with supportive evidence from newspapers. These display fundamental concepts among the English poor, who were often poorly literate, and who comprised the majority of the population. Discussion focuses upon their understandings of 'home', 'belonging', 'friends', and 'community'. These key concepts are related here to modern discussions, to set important concerns into historical perspective. 'Friends', valuably studied by sociologists such as Pahl, had a wide meaning in the past. 'Home' meant (alongside abode) one's parish of legal settlement, where one was entitled to poor relief under the settlement/poor laws. This was where one 'belonged'. Ideas of 'community' were held and displayed even at a distance, among frequently migrant poor, who wrote to their parishes showing strong ties of attachment, right, and local obligation. This discussion explores these issues in connection with belonging and identity. It elucidates the meaning and working of poor law settlement, and is also an exploration of popular mentalities and the semi-literate ways in which these were expressed.

Class I and III polyhydroxyalkanoate synthases from Ralstonia eutropha and Allochromatium vinosum: characterization and substrate specificity studies.

Class I and III polyhydroxyalkanoate (PHA) synthases catalyze the conversion of beta-hydroxybutyryl coenzyme A (HBCoA) to polyhydroxybutyrate. The Class I PHA synthase from Ralstonia eutropha has been purified by numerous labs with reported specific activities that vary between 1 and 160 U/mg. An N-terminal (His)6-PHA synthase was constructed and purified with specific activity of 40 U/mg. The variable activity is shown to be related to the protein's propensity to aggregate and not to incomplete post-translational modification by coenzyme A and a phosphopantetheinyl transferase. The substrate specificities of this enzyme and the Class III PHA synthase from Allochromatium vinosum have been determined with nine analogs of varied chain length and branching, OH group position within the chain, and thioesters. The results suggest that in vitro, both PHA synthases are very specific and provide further support for their active site structural similarities. In vitro results differ from studies in vivo.

PHA synthase activity controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo.

A synthetic operon for polyhydroxyalkanoate (PHA) biosynthesis designed to yield high levels of PHA synthase activity in vivo was constructed by positioning a genetic fragment encoding beta-ketothiolase and acetoacetyl-CoA reductase behind a modified synthase gene containing an Escherichia coli promoter and ribosome binding site. Plasmids containing the synthetic operon and the native Alcaligenes eutrophus PHA operon were transformed into E. coli DH5 alpha and analyzed for polyhydroxybutyrate production. The molecular weight of polymer isolated from recombinant E. coli containing the modified synthase construct, determined by multiangle light scattering, was lower than that of the polymer from E. coli containing the native A. eutrophus operon. A further decrease in polyester molecular weight was observed with increased induction of the PHA biosynthetic genes in the synthetic operon. Comparison of the enzyme activity levels of PHA biosynthetic enzymes in a strain encoding the native operon with a strain possessing the synthetic operon indicates that the amount of polyhydroxyalkanoate synthase in a host organism plays a key role in controlling the molecular weight and the polydispersity of polymer.

Overexpression and purification of the soluble polyhydroxyalkanoate synthase from Alcaligenes eutrophus: evidence for a required posttranslational modification for catalytic activity.

Polyhydroxyalkanoate (PHA) synthase has been expressed in Escherichia coli by reengineering the 5'-end of the wild-type (wt) gene and subsequent transformation of this gene into protease-deficient E. coli UT5600 (ompT-). Induction with IPTG results in soluble PHA synthase, which is approximately 5% of the total protein. The soluble synthase has been purified to > 90% homogeneity using FPLC chromatography on hydroxylapatite and Q-Sepharose and has a specific activity of 5 mumol min-1 mg-1. The molecular weight of the PHA product is approximately 10(6) Da based on PlGel chromatography and calibration using polystyrene molecular weight markers. The synthase in the absence of substrate appears to exist in both monomeric and dimeric forms. Incubation of the synthase with an excess of substrate converts it into a form that is now extractable into CHCl3 and sediments on sucrose density ultracentrifugation with PHA. Studies in which the ratio of substrate, 3-D-hydroxybutyrylCoA, to synthase is varied suggest that during polymerization the elongation process occurs at a rate much faster than during the initiation process. A mechanistic model has been proposed for the polymerization process [Griebel, R., Smith, Z., & Merrick, J. (1968) Biochemistry 7, 3676-3681] in which two cysteines are required for catalysis. This model is based on the well-characterized enzymes involved in fatty acid biosynthesis. To test this model, several site-directed mutants of synthase, selected based on sequence conservation among synthases, have been prepared. The C459S mutant has activity approximately 90% that of the wt protein, while the C319S and C319A synthases possess < 0.01% the activity of the wt protein. CD and antibody studies suggest that the mutant proteins are properly folded. The detection of only a single essential cysteine by mutagenesis and the requirement for posttranslational modification by phosphopantetheine to provide a second thiol in many enzymes utilizing coenzyme A thiol ester substrates made us consider the possibility that posttranslational modification was required for synthase activity as well. This hypothesis was confirmed when the plasmid containing PHA synthase (pKAS4) was transformed into E. coli SJ16, requiring beta-alanine for growth. Growth of SJ16/pKAS4 on [3H]-beta-alanine followed by Coomassie staining of the protein and autoradiography revealed that PHA synthase is overexpressed and that beta-alanine is incorporated into the protein. These results suggest PHA synthase is posttranslationally modified by phosphopantetheine.(ABSTRACT TRUNCATED AT 400 WORDS)