Colonization of heterochromatic genes by transposable elements in Drosophila

As a further step toward understanding transposable element-host genome interactions, we investigated the molecular anatomy of introns from five heterochromatic and 22 euchromatic protein-coding genes of Drosophila melanogaster. A total of 79 kb of intronic sequences from heterochromatic genes and 355 kb of intronic sequences from euchromatic genes have been used in Blast searches against Drosophila transposable elements (TEs). The results show that TE-homologous sequences belonging to 19 different families represent about 50% of intronic DNA from heterochromatic genes. In contrast, only 0.1% of the euchromatic intron DNA exhibits homology to known TEs. Intraspecific and interspecific size polymorphisms of introns were found, which are likely to be associated with changes in TE-related sequences. Together, the enrichment in TEs and the apparent dynamic state of heterochromatic introns suggest that TEs contribute significantly to the evolution of genes located in heterochromatin.     

Surface expression of inward rectifier potassium channels is controlled by selective Golgi export

Traffic of integral membrane proteins along the secretory pathway is not simply a default process but can be selective. Such selectivity is achieved by sequence information within the cargo protein that recruits coat protein complexes to drive the formation of transport vesicles. A number of sequence motifs have been identified in the cytoplasmic domains of ion channels that regulate early trafficking events between the endoplasmic reticulum and the Golgi complex. Here, we demonstrate that the following trafficking step from the Golgi compartment to the plasma membrane can also be selective. The N-terminal domain of the inward rectifier potassium channel Kir2.1 contains specific sequence information that is necessary for its efficient export from the Golgi complex. Lack of this information results in accumulation of the protein within the Golgi and a significant decrease in cell surface expression. As similar results were obtained for the N terminus of another Kir channel subfamily member, Kir4.1, which could functionally substitute for the Kir2.1 N terminus, we propose a more general role of the identified N-terminal domains for post-Golgi trafficking of Kir channels.     

Tumor tissue concentrations of the proteinase inhibitors tissue inhibitor of metalloproteinases-1 (TIMP-1) and plasminogen activator inhibitor type 1 (PAI-1) are complementary in determining prognosis in primary breast cancer

The purpose of this study was to investigate the association between tumor tissue levels of total tissue inhibitor of metalloproteinases-1 (TIMP-1) and prognosis in patients with primary breast cancer and to analyze whether measurement of TIMP-1 in tumor extracts added prognostic information to that obtained from measurements of urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 (PAI-1). An established sandwich enzyme-linked immunosorbent assay was thoroughly validated for the measurement of total TIMP-1 in tumor tissue extracts and used to determine levels of total TIMP-1 in 341 detergent-extracted tumor tissue samples from patients with primary breast cancer. The median age of the patients was 56 years (range, 29-75 years), and 164 were lymph node-negative, and 177 were lymph node-positive. The median follow-up time of the patients was 8.5 years (range, 7.3-11.3 years), and during follow-up 153 patients experienced recurrence of disease, and 136 patients died. In univariate survival analysis, we found a significant association between tumor tissue TIMP-1 level and both shorter recurrence-free survival (p = 0.0004) and shorter overall survival (p = 0.03). In multivariate survival analysis, higher tumor tissue TIMP-1 levels significantly and independently predicted shorter recurrence-free survival (p < 0.05, hazard ratios >1, comparing quartiles II-IV with I). In addition, we found that measurement of TIMP-1 levels added prognostic information to that obtained from measurement of PAI-1. In conclusion, high levels of TIMP-1 in tumor tissue extracts are significantly associated with a poor prognosis in patients with primary breast cancer. Furthermore TIMP-1 adds prognostic information to that obtained from PAI-1. However, further validation in independent data sets is needed.     

Selective interactions between vertebrate polycomb homologs and the SUV39H1 histone lysine methyltransferase suggest that histone H3-K9 methylation contributes to chromosomal targeting of Polycomb group proteins

Polycomb group (PcG) proteins form multimeric chromatin-associated protein complexes that are involved in heritable repression of gene activity. Two distinct human PcG complexes have been characterized. The EED/EZH2 PcG complex utilizes histone deacetylation to repress gene activity. The HPC/HPH PcG complex contains the HPH, RING1, BMI1, and HPC proteins. Here we show that vertebrate Polycomb homologs HPC2 and XPc2, but not M33/MPc1, interact with the histone lysine methyltransferase (HMTase) SUV39H1 both in vitro and in vivo. We further find that overexpression of SUV39H1 induces selective nuclear relocalization of HPC/HPH PcG proteins but not of the EED/EZH2 PcG proteins. This SUV39H1-dependent relocalization concentrates the HPC/HPH PcG proteins to the large pericentromeric heterochromatin domains (1q12) on human chromosome 1. Within these PcG domains we observe increased H3-K9 methylation. Finally, we show that H3-K9 HMTase activity is associated with endogenous HPC2. Our findings suggest a role for the SUV39H1 HMTase and histone H3-K9 methylation in the targeting of human HPC/HPH PcG proteins to modified chromatin structures.     

The Erv41p-Erv46p complex: multiple export signals are required in trans for COPII-dependent transport from the ER

Erv41p and Erv46p form an integral membrane protein complex that cycles between the endoplasmic reticulum (ER) and Golgi. Both proteins contain a large lumenal domain and short N- and C-terminal tail sequences exposed to the cytosol. The coat protein complex II (COPII) packages the Erv41p-Erv46p complex into ER-derived vesicles for delivery to the Golgi. We determined signals in the Erv41p-Erv46p complex that are required for COPII-dependent export from the ER. Mutants lacking the Erv41p or Erv46p C-terminus accumulated in the ER and were not packaged efficiently into vesicles. We identified an isoleucine-leucine sequence in the Erv41p tail that was required for COPII binding and inclusion of the complex into vesicles. This signal was sufficient for COPII binding but not for ER export. The Erv46p tail contains a phenylalanine-tyrosine sequence required together with the isoleucine-leucine signal in Erv41p for export of the complex. Surprisingly, Erv41p- Erv46p tail-swapped chimeras were not exported from the ER, indicating that signals in both the Erv41p and the Erv46p tail sequences are required in a specific orientation for efficient packaging of the Erv41p-Erv46p complex.     

Identification of the major chlorosomal bacteriochlorophylls of the green sulfur bacteria Chlorobium vibrioforme and Chlorobium phaeovibrioides; their function in lateral energy transfer

The chlorosomal bacteriochlorophyll (BChl) composition of the green sulfur bacteria Chlorobium vibrioforme and Chlorobium phaeovibrioides was investigated by means of normal-phase high-performance liquid chromatography. From both species a number of homologues was isolated, which were identified by absorption and ²⁵²Cf-plasma desorption mass spectroscopy. Besides BChl d, C. vibrioforme contained a significant amount of BChl c, which may provide an explanation for the previous observation of at least two spectrally different pools of BChl in the chlorosomes of green sulfur bacteria (Otte et al. 1991). C. phaeovibrioides contained various homologues of BChl e only. Absorption spectra in acetone of BChl c, d and e, as well as bacteriopheophytin e are presented. No systematic differences were found for the various homologues of each pigment. In addition to farnesol, the mass spectra revealed the presence of various minor esterifying alcohols in both species, including phytol, oleol, cetol and 4-undecyl-2-furanmethanol, as well as an alcohol of low molecular mass, which is tentatively assumed to be decenol.Abbreviations BChl bacteriochlorophyll - BPh bacteriopheophytin (used as a general name for the Mg-free compound, irrespective of the esterifying alcohol) - HPLC high-performance liquid chromatography     

Reversible hydrogenase in Anabaena variabilis ATCC 29413

The presence and localization of a reversible hydrogenase in non-N₂-fixing cells of the filamentous cyanobacterium Anabaena variabilis were investigated by in vitro activity measurements, native-PAGE/activity stain, SDS-PAGE/Western immunoblots, and immunogold localization. Reversible hydrogenase activity was induced approximately 100-fold by sparging the cell suspensions with a mixture of 99% argon and 1% CO₂ for 20–26 h. Native-PAGE/activity stain demonstrated the presence of an in vitro functional enzyme with an apparent molecular mass of 118 kDa. Native-PAGE/Western immunoblots, using polyclonal antisera directed against purified hydrogenase from the purple sulphur bacterium Thiocapsa roseopersicina, detected two native proteins with molecular masses of 118 and 133 kDa, respectively. SDS-PAGE/Western immunoblots confirmed the presence of a single polypeptide with a molecular mass of approximately 40 kDa in both induced and non-induced cells. Immunocytolocalization experiments using ultrathin sections again demonstrated the presence of hydrogenase in both induced and non-induced cells. A higher specific labeling was associated with the thylakoid regions, which, using an image analyzer, was calculated to be approximately 4 x higher per cell area compared to in the centroplasm. It is suggested that anaerobic incubation induces higher reversible hydrogenase activity, regulated mainly at the level of activating (pre)existing form(s) of inactive enzyme(s)/protein(s), maybe in combination with synthesis of additional subunit(s).     

Energy conservation in malolactic fermentation by Lactobacillus plantarum and Lactobacillus sake

A comparably poor growth medium containing 0.1% yeast extract as sole non-defined constituent was developed which allowed good reproducible growth of lactic acid bacteria. Of seven different strains of lactic acid bacteria tested, only Lactobacillus plantarum and Lactobacillus sake were found to catalyze stoichiometric conversion of l-malate to l-lactate and CO₂ concomitant with growth. The specific growth yield of malate fermentation to lactate at pH 5.0 was 2.0 g and 3.7 g per mol with L. plantarum and L. sake, respectively. Growth in batch cultures depended linearly on the malate concentration provided. Malate was decarboxylated nearly exclusively by the cytoplasmically localized malo-lactic enzyme. No other C₄-dicarboxylic acid-decarboxylating enzyme activity could be detected at significant activity in cell-free extracts. In pH-controlled continuous cultures, L. plantarum grew well with glucose as substrate, but not with malate. Addition of lactate to continuous cultures metabolizing glucose or malate decreased cell yields significantly. These results indicate that malo-lactic fermentation by these bacteria can be coupled with energy conservation, and that membrane energetization and ATP synthesis through this metabolic activity are due to malate uptake and/or lactate excretion rather than to an ion-translocating decarboxylase enzyme.