Evidence for protein phosphorylation inStreptomyces lincolnensis

Protein phosphorylation was investigated inStreptomyces lincolnensis underin vivo conditions. In cells grown in the presence of³²P-orthophosphate, proteins ofM=12, 22, 45, 68 and 90 kDa were labeled with³²P (detected by gel electrophoresis and autoradiography). These proteins were shown to contain O-phosphoserine and a small proportion of O-phosphotyrosine. Taken together the results indicate thatStreptomyces lincolnensis harbors several protein kinases including a protein-tyrosine kinase activity.     

Intraspecific nuclear DNA variation in Drosophila

We have summarized and analyzed all available nuclear DNA sequence polymorphism studies for three species of Drosophila, D. melanogaster (24 loci), D. simulans (12 loci), and D. pseudoobscura (5 loci). Our major findings are: (1) The average nucleotide heterozygosity ranges from about 0.4% to 2% depending upon species and function of the region, i.e., coding or noncoding. (2) Compared to D. simulans and D. pseudoobscura (which are about equally variable), D. melanogaster displays a low degree of DNA polymorphism. (3) Noncoding introns and 3' and 5' flanking DNA shows less polymorphism than silent sites within coding DNA. (4) X-linked genes are less variable than autosomal genes. (5) Transition (Ts) and transversion (Tv) polymorphisms are about equally frequent in non-coding DNA and at fourfold degenerate sites in coding DNA while Ts polymorphisms outnumber Tv polymorphisms by about 2:1 in total coding DNA. The increased Ts polymorphism in coding regions is likely due to the structure of the genetic code: silent changes are more often Ts's than are replacement substitutions. (6) The proportion of replacement polymorphisms is significantly higher in D. melanogaster than in D. simulans. (7) The level of variation in coding DNA and the adjacent noncoding DNA is significantly correlated indicating regional effects, most notably recombination. (8) Surprisingly, the level of polymorphism at silent coding sites in D. melanogaster is positively correlated with degree of codon usage bias. (9) Three proposed tests of the neutral theory of DNA polymorphisms have been performed on the data: Tajima's test, the HKA test, and the McDonald-Kreitman test. About half of the loci fail to conform to the expectations of neutral theory by one of the tests. We conclude that many variables are affecting levels of DNA polymorphism in Drosophila, from properties of nucleotides to population history and, perhaps, mating structure. No simple, all encompassing explanation satisfactorily accounts for the data.      

A molecular phylogeny for the Drosophila melanogaster subgroup and the problem of polymorphism data

Drosophila melanogaster belongs to a closely related group of eight species collectively known as the melanogaster subgroup; all are native to sub-Saharan Africa and islands off the east coast of Africa. The phylogenetic relationships of most species in this subgroup have been well documented; however, the three most closely related species, D. simulans, D. sechellia, and D. mauritiana, have remained problematic from a phylogenetic standpoint as no data set has unambiguously resolved them. We present new DNA sequence data on the nullo and Serendipity-alpha genes and combine them with all available nuclear DNA sequence data; the total data encompass 12 genes and the ITS of rDNA. A methodological problem arose because nine of the genes had information on intraspecific polymorphisms in at least one species. We explored the effect of inclusion/exclusion of polymorphic sites and found that it had very little effect on phylogenetic inferences, due largely to the fact that 82% of polymorphisms are autapomorphies (unique to one species). We have also reanalyzed our previous DNA-DNA hybridization data with a bootstrap procedure. The combined sequence data set and the DNA-DNA hybridization data strongly support the sister status of the two island species, D. sechellia and D. mauritiana. This at least partially resolves what had been a paradox of parallel evolution in these two species.      

Structural properties of potexvirus coat proteins detected by optical methods

It has been shown by X-ray analysis that cores of coat proteins (CPs) from three potexviruses, flexible helical RNA-containing plant viruses, have similar α-helical structure. However, this similarity cannot explain structural lability of potexvirus virions, which is believed to determine their biological activity. Here, we used circular dichroism (CD) spectroscopy in the far UV region to compare optical properties of CPs from three potexviruses with the same morphology and similar structure. CPs from Alternanthera mosaic virus (AltMV), potato aucuba mosaic virus (PAMV), and potato virus X (PVX) have been studied in a free state and in virions. The CD spectrum of AltMV virions was similar to the previously obtained CD spectrum of papaya mosaic virus (PapMV) virions, but differed significantly from the CD spectrum of PAMV virions. The CD spectrum of PAMV virions resembled in its basic characteristics the CD spectrum of PVX virions characterized by molar ellipticity that is abnormally low for α-helical proteins. Homology modeling of the CP structures in AltMV, PAMV, and PVX virions was based on the known high-resolution structures of CPs from papaya mosaic virus and bamboo mosaic virus and confirmed that the structures of the CP cores in all three viruses were nearly identical. Comparison of amino acid sequences of different potexvirus CPs and prediction of unstructured regions in these proteins revealed a possible correlation between specific features in the virion CD spectra and the presence of disordered N-terminal segments in the CPs.     

Influence of the cystic fibrosis transmembrane conductance regulator on expression of lipid metabolism-related genes in dendritic cells

Background

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Infections of the respiratory tract are a hallmark in CF. The host immune responses in CF are not adequate to eradicate pathogens, such as P. aeruginosa. Dendritic cells (DC) are crucial in initiation and regulation of immune responses. Changes in DC function could contribute to abnormal immune responses on multiple levels. The role of DC in CF lung disease remains unknown.

Methods

This study investigated the expression of CFTR gene in bone marrow-derived DC. We compared the differentiation and maturation profile of DC from CF and wild type (WT) mice. We analyzed the gene expression levels in DC from naive CF and WT mice or following P. aeruginosa infection.

Results

CFTR is expressed in DC with lower level compared to lung tissue. DC from CF mice showed a delayed in the early phase of differentiation. Gene expression analysis in DC generated from naive CF and WT mice revealed decreased expression of Caveolin-1 (Cav1), a membrane lipid raft protein, in the CF DC compared to WT DC. Consistently, protein and activity levels of the sterol regulatory element binding protein (SREBP), a negative regulator of Cav1 expression, were increased in CF DC. Following exposure to P. aeruginosa, expression of 3β-hydroxysterol-Δ7 reductase (Dhcr7) and stearoyl-CoA desaturase 2 (Scd2), two enzymes involved in the lipid metabolism that are also regulated by SREBP, was less decreased in the CF DC compared to WT DC.

Conclusion

These results suggest that CFTR dysfunction in DC affects factors involved in membrane structure and lipid-metabolism, which may contribute to the abnormal inflammatory and immune response characteristic of CF.     

Low cetyltrimethylammonium bromide concentrations induce reversible amorphous aggregation of tobacco mosaic virus and its coat protein at room temperature

Ordered and amorphous protein aggregation causes numerous diseases. Tobacco mosaic virus coat protein for many decades serves as the classical model of ordered protein aggregation ("polymerization"). It was also found to be highly prone to heat-induced amorphous aggregation and the rate of this aggregation could be easily manipulated by changes in solution ionic strength and temperature. Here, we report that rapid amorphous aggregation of this protein can be induced at 25 degrees C in phosphate buffer by low micromolar (start at about 15 microM) concentrations of cationic surfactant cetyltrimethylammonium bromide. At equilibrium four surfactant molecules bound to the protein subunit. As judged by circular dichroism and fluorescence spectroscopy data, the coat protein molecules retained their native structure upon the cetyltrimethylammonium bromide induced aggregation. No aggregation was observed at the higher surfactant concentrations (above 300 microM). Micromolar concentrations of anionic surfactant sodium dodecylsulfate rapidly reversed the cetyltrimethylammonium bromide induced aggregation of the coat protein due to formation of mixed surfactant-surfactant micelles. Cetyltrimethylammonium bromide (100-300 microM) also induced the reversible intact tobacco mosaic virus virion aggregation. The possible liability to the cetyltrimethylammonium bromide induced amorphous aggregation of other ordered aggregate-producing proteins has been discussed.