Molecular evolution of P transposable elements in the genus Drosophila. III. The melanogaster species group

Phylogenetic relationships were determined for 76 partial P-element sequences from 14 species of the melanogaster species group within the Drosophila subgenus Sophophora. These results are examined in the context of the phylogeny of the species from which the sequences were isolated. Sequences from the P-element family fall into distinct subfamilies, or clades, which are often characteristic for particular species subgroups. When examined locally among closely related species, the evolution of P elements is characterized by vertical transmission, whereby the P-element phylogeny traces the species phylogeny. On a broader scale, however, the P-element phylogeny is not congruent with the species phylogeny. One feature of P-element evolution in the melanogaster group is the presence of more than one P-element subfamily, differing by as much as 36%, in the genomes of some species. Thus, P elements from several individual species are not monophyletic, and a likely explanation for the incongruence between P-element and species phylogenies is provided by the comparison of paralogous sequences. In certain instances, horizontal transfer seems to be a valid alternative explanation for lack of congruence between species and P-element phylogenies. The canonical P-element subfamily, which represents the active, autonomous transposable element, is restricted to D. melanogaster. Thus, its origin clearly lies outside of the melanogaster species group, consistent with the earlier conclusion of recent horizontal transfer.      

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Noisy-threshold control of cell death

Background  

Cellular responses to death-promoting stimuli typically proceed through a differentiated multistage process, involving a lag phase, extensive death, and potential adaptation. Deregulation of this chain of events is at the root of many diseases. Improper adaptation is particularly important because it allows cell sub-populations to survive even in the continuous presence of death conditions, which results, among others, in the eventual failure of many targeted anticancer therapies.     

Fishing for biodiversity: novel methanopterin-linked C transfer genes deduced from the Sargasso Sea metagenome

The recently generated database of microbial genes from an oligotrophic environment populated by a calculated 1800 major phylotypes (the Sargasso Sea metagenome-SSM) presents a great source for expanding local databases of genes indicative of a specific function. In this article we analyse the SSM for the presence of methanopterin-linked C1 transfer genes that are signature for methylotrophy. We conclude that more than 10 phylotypes possessing genes of interest are present in this environment. The sequences representative of these major phylotypes do not appear to belong to any known microbial group capable of methanopterin-linked C1 transfer. Instead, these sequences separate from all known sequences on phylogenetic trees, pointing toward their affiliation with novel microbial phyla. These data imply a broader distribution of methanopterin-linked functions in the microbial world than has been previously known.     

Development and application of polymerase chain reaction primers based on fhcD for environmental detection of methanopterin-linked C1-metabolism in bacteria

In this work we describe development and testing of a novel pair of environmental primers targeting fhcD, a conserved gene in the H4MTP-linked C1-transfer pathway, and demonstrate that these primers enable confident detection of a broad variety of fhcD genes originating from phylogenetically diverse bacteria. The new primer pair was employed to analyse fhcD diversity in Lake Washington sediment, uncovering the presence of 40 fhcD phylotypes. Based on phylogenetic analyses, the phylotypes identified were affiliated with alpha-, beta- and gamma-proteobacteria, and Planctomycetes, while a number of sequences formed deep branches suggesting the presence of unknown groups of microorganisms. To assess the physiological potential and the possible substrate repertoire of the fhcD-containing species in Lake Washington, we conducted enrichments of natural populations on a variety of C1 substrates, and observed specific shifts in community structure in response to different C1 substrates. A specific shift in community structure was also observed in the presence of humic acids suggesting that C1 transfer metabolism linked to H4MPT may be part of the degradation pathway for this natural polymer, possibly involving formaldehyde production. Overall, our data suggest that C1 oxidation reactions linked to H4MPT are much more widespread in natural environments than previously thought.     

MtdC, a novel class of methylene tetrahydromethanopterin dehydrogenases

Novel methylene tetrahydromethanopterin (H4MPT) dehydrogenase enzymes, named MtdC, were purified after expressing in Escherichia coli genes from, respectively, Gemmata sp. strain Wa1-1 and environmental DNA originating from unidentified microbial species. The MtdC enzymes were shown to possess high affinities for methylene-H4MPT and NADP but low affinities for methylene tetrahydrofolate or NAD. The substrate range and the kinetic properties revealed by MtdC enzymes distinguish them from the previously characterized bacterial methylene-H4MPT dehydrogenases, MtdA and MtdB. While revealing higher sequence similarity to MtdA enzymes, MtdC enzymes appear to fulfill a function homologous to the function of MtdB, as part of the H4MPT-linked pathway for formaldehyde oxidation/detoxification.