Ackr3-Venus knock-in mouse lights up brain vasculature

The atypical chemokine receptor 3, ACKR3, is a G protein-coupled receptor, which does not couple to G proteins but recruits βarrestins. At present, ACKR3 is considered a target for cancer and cardiovascular disorders, but less is known about the potential of ACKR3 as a target for brain disease. Further, mouse lines have been created to identify cells expressing the receptor, but there is no tool to visualize and study the receptor itself under physiological conditions. Here, we engineered a knock-in (KI) mouse expressing a functional ACKR3-Venus fusion protein to directly detect the receptor, particularly in the adult brain. In HEK-293 cells, native and fused receptors showed similar membrane expression, ligand induced trafficking and signaling profiles, indicating that the Venus fusion does not alter receptor signaling. We also found that ACKR3-Venus enables direct real-time monitoring of receptor trafficking using resonance energy transfer. In ACKR3-Venus knock-in mice, we found normal ACKR3 mRNA levels in the brain, suggesting intact gene transcription. We fully mapped receptor expression across 14 peripheral organs and 112 brain areas and found that ACKR3 is primarily localized to the vasculature in these tissues. In the periphery, receptor distribution aligns with previous reports. In the brain there is notable ACKR3 expression in endothelial vascular cells, hippocampal GABAergic interneurons and neuroblast neighboring cells. In conclusion, we have generated Ackr3-Venus knock-in mice with a traceable ACKR3 receptor, which will be a useful tool to the research community for interrogations about ACKR3 biology and related diseases.

Impacts of allopolyploidization and structural variation on intraspecific diversification in Brassica rapa

Background Despite the prevalence and recurrence of polyploidization in the speciation of flowering plants, its impacts on crop intraspecific genome diversification are largely unknown. Brassica rapa is a mesopolyploid species that is domesticated into many subspecies with distinctive morphotypes. Results Herein, we report the consequences of the whole-genome triplication (WGT) on intraspecific diversification using a pan-genome analysis of 16 de novo assembled and two reported genomes. Among the genes that derive from WGT, 13.42% of polyploidy-derived genes accumulate more transposable elements and non-synonymous mutations than other genes during individual genome evolution. We denote such genes as being “flexible.” We construct the Brassica rapa ancestral genome and observe the continuing influence of the dominant subgenome on intraspecific diversification in B. rapa. The gene flexibility is biased to the more fractionated subgenomes (MFs), in contrast to the more intact gene content of the dominant LF (least fractionated) subgenome. Furthermore, polyploidy-derived flexible syntenic genes are implicated in the response to stimulus and the phytohormone auxin; this may reflect adaptation to the environment. Using an integrated graph-based genome, we investigate the structural variation (SV) landscapes in 524 B. rapa genomes. We observe that SVs track morphotype domestication. Four out of 266 candidate genes for Chinese cabbage domestication are speculated to be involved in the leafy head formation. Conclusions This pan-genome uncovers the possible contributions of allopolyploidization on intraspecific diversification and the possible and underexplored role of SVs in favorable trait domestication. Collectively, our work serves as a rich resource for genome-based B. rapa improvement.

Desert hedgehog knockout mice show a reduced ratio of CD4+:CD8+ in thymic developing T cells

<p class="abstract"><strong>Background:</strong> Thymic differentiation is important and determines the strength of adaptive immunity in mammals in their later days in life. There are many factors that have been found to influence the development of T lymphocytes in the thymus and these include the effect of hedgehog signalling family of proteins. Immunologists and other basic science researchers have established the role of Indian hedgehog and sonic hedgehog in thymocytes development in the recent past, but the role of hedgehog is not well known. The aim of this study was to determine the influence of Desert hedgehog in CD4:CD8 ratio in developing thymocytes of mice.</p><p class="abstract"><strong>Methods:</strong> Smashed thymocytes from mice deficient of Desert hedgehog and those with an intact gene coding for this protein were prepared in a cell suspension using standard procedurs. The cell suspensions were stained using fluorochrome-labelled monoclonal anti: CD4-PE, CD8-TRI, CD3-FITC, CD5-FITC, CD44-PE and CD25-FITC (e-Bioscience). Samples were analyzed using a three-color flow cytometry. The flow cytometry-generated data was analyzed using flowjo (Treestar, USA). Statistical significance of the findings was determined using paired t-test and reported at p&lt;0.05. </p><p class="abstract"><strong>Results:</strong> There was a general upward trend on CD4+CD8+ double positive thymocytes in Desert hedgehog mice relative to WT controls. An analysis of CD4:8 ratio revealed a reduced ratio in Dhh KO mice compared to that of WT controls attributable to the finding that there might have been a preferential differentiation of DP CD4+CD8+ to SP CD4+ in Dhh knockout mice as demonstrated by percentage of thymic subsets. The results of this study were not statistically significant and this was blamed on the fewer number of animals used in the study.</p><p class="abstract"><strong>Conclusions:</strong> Dhh might have a role arresting the DP cell subjects from differentiating preferentially to CD4+ T cells. To get statistically significant findings, these experiments could be repeated with a larger animal sample.</p>

Comparison of lipooligosaccharide biosynthesis genes of Campylobacter jejuni strains with varying abilities to colonize the chicken gut and to invade Caco-2 cells

Campylobacter jejuni strains develop a high variability of lipooligosaccharide (LOS) structures on the cell surface based on variations in the genetic content of the LOS biosynthesis locus. While the importance of these variations for ganglioside mimicry as a critical factor in the triggering of Guillain–Barré syndrome has already been shown, little work has been done on the investigation of LOS structures and their function in the pathogenesis of gastrointestinal disease. In this study, the presence of several LOS genes in 40 C. jejuni strains with different abilities to colonize the chicken gut and to invade Caco-2 cells was investigated by PCR. Two genes, cgtB and wlaN, encoding putative β-1,3-galactosyltransferases were detected in most strongly invasive strains and rarely in non-invasive strains. A homopolymeric tract within the wlaN gene resulted in an intact gene product only in strongly invasive strains. The specific function of these genes during LOS biosynthesis is still unknown. cgtB and wlaN gene products are suggested to be involved in development of the colonization and invasion ability of C. jejuni. After a classification of the complete LOS loci, an association between a particular LOS class and colonization and invasion ability of the C. jejuni strain could not be detected. Lack of the pglB gene involved in protein glycosylation in one strain could be responsible for the weak colonization and invasion ability of this strain. There is some evidence that different genetic characteristics were responsible for strong or weak colonization and the invasion ability of C. jejuni strains.

Shuttle mutagenesis and targeted disruption of a telomere-located essential gene of Leishmania

Leishmania mutants have contributed greatly to extend our knowledge of this parasite's biology. Here we report the use of the mariner in vitro transposition system as a source of reagents for shuttle mutagenesis and targeted disruption of Leishmania genes. The locus-specific integration was achieved by the disruption of the subtelomeric gene encoding a DNA-directed RNA polymerase III subunit (RPC2). Further inactivation of RPC2 alleles required the complementation of the intact gene, which was transfected in an episomal context. However, attempts to generate a RPC2 chromosomal null mutant resulted in genomic rearrangements that maintained copies of the intact locus in the genome. The maintenance of the RPC2 chromosomal locus in complemented mutants was not mediated by an increase in the number of copies and did not involve chromosomal translocations, which are the typical characteristics of the genomic plasticity of this parasite. Unlike the endogenous locus, the selectable marker used to disrupt RPC2 did not display a tendency to remain in its chromosomal location but was targeted into supernumerary episomal molecules.

Identification of a Tomatinase in the Tomato-Pathogenic Actinomycete Clavibacter michiganensis subsp. michiganensis NCPPB382

The insertion site of a transposon mutant of Clavibacter michiganensis subsp. michiganensis NCPPB382 was cloned and found to be located in the gene tomA encoding a member of the glycosyl hydrolase family 10. The intact gene was obtained from a cosmid library of C. michiganensis subsp. michiganensis. The deduced protein TomA (543 amino acids, 58 kDa) contains a predicted signal peptide and two domains, the N-terminal catalytic domain and a C-terminal fibronectin III-like domain. The closest well-characterized relatives of TomA were tomatinases from fungi involved in the detoxification of the tomato saponin α-tomatine which acts as a growth inhibitor. Growth inhibition of C. michiganensis subsp. michiganensis by α-tomatine was stronger in the tomA mutants than in the wild type. Tomatinase activity assayed by deglycosylation of α-tomatine to tomatidine was demonstrated in concentrated culture supernatants of C. michiganensis subsp. michiganensis. No activity was found with the tomA mutants. However, neither the transposon mutant nor a second mutant constructed by gene disruption was affected in virulence on the tomato cv. Moneymaker.

The Stress-Responsive dgk Gene from Streptococcus mutans Encodes a Putative Undecaprenol Kinase Activity

ABSTRACT We analyzed a previously constructed stress-sensitive Streptococcus mutans mutant Tn-1 strain resulting from disruption by transposon Tn916 of a gene encoding a protein exhibiting amino acid sequence similarity to the Escherichia coli diacylglycerol kinase. It was confirmed that the mutation led to significantly reduced lipid kinase activity, while expression of the intact gene on a plasmid restored both kinase activity and the wild-type phenotype. Further analysis revealed that the product of the dgk gene in S. mutans predominantly recognizes a lipid substrate other than diacylglycerol, most likely undecaprenol, as demonstrated by its efficient phosphorylation and the resistance of the product of the reaction to saponification. The physiological role of the product of the dgk gene as a putative undecaprenol kinase was further supported by a significantly higher sensitivity of the mutant to bacitracin compared with that of the parental strain.

A point mutation in the UDP-glucose pyrophosphorylase gene results in decreases of UDP-glucose and inactivation of glycogen synthase

The regulatory role of UDP-glucose in glycogen biogenesis was investigated in fibroblasts containing a point mutation in the UDP-glucose pyrophosphorylase gene and, consequently, chronically low UDP-glucose levels (Qc). Comparisons were made with cells having the intact gene and restored UDP-glucose levels (G3). Glycogen was always very low in Qc cells. [14C]Glucose incorporation into glycogen was decreased and unaffected by insulin in Qc cells, whereas insulin stimulated glucose incorporation by 50% in G3 cells. Glycogen synthase (GS) activity measured in vitro was virtually absent and the amount of enzyme in Qc cells was decreased by about 50%. The difference in GS activity between cells persisted even when G3 cells were devoid of glycogen. Incubation of G3 cell extracts with either exogenous UDP-glucose or glycogen resulted in increases in GS activity. Incubation of Qc cell extracts with exogenous UDP-glucose had no effect on GS activity; however, incubation with glycogen fully restored enzyme activity. Incubation of G3 cell extracts with radioactive UDP-glucose resulted in substantial binding of ligand to immunoprecipitated GS, whereas no binding was detected in Qc immunoprecipitates. Incubation of Qc cell extracts with exogenous glycogen fully restored UDP-glucose binding in the immunoprecipitate. These data suggest that chronically low UDP-glucose levels in cells result in inactivation of GS, owing to loss of the ability of GS to bind UDP-glucose.

Purification and Characterization of a UDP-Glucosyltransferase Produced by Legionella pneumophila

ABSTRACT Legionella pneumophila is the agent of Legionnaires' disease. It invades and replicates within eukaryotic cells, including aquatic protozoans, mammalian macrophages, and epithelial cells. The molecular mechanisms of the Legionella interaction with target cells are not fully defined. In an attempt to discover novel virulence factors of L. pneumophila, we searched for bacterial enzymes with transferase activity. Upon screening ultrasonic extracts of virulent legionellae, we identified a uridine diphospho (UDP)-glucosyltransferase activity, which was capable of modifying a 45-kDa substrate in host cells. An approximately 60-kDa UDP-glucosyltransferase was purified from L. pneumophila and subjected to microsequencing. An N-terminal amino acid sequence, as well as the sequence of an internal peptide, allowed us to identify the gene for the enzyme within the unfinished L. pneumophila genome database. The intact gene was cloned and expressed in Escherichia coli, and the recombinant protein was purified and confirmed to possess an enzymatic activity similar to that of the native UDP-glucosyltransferase. We designated this gene ugt (UDP-glucosyltransferase). The Legionella enzyme did not exhibit significant homology with any known protein, suggesting that it is novel in structure and, perhaps, in function. Based on PCR data, an enzyme assay, and an immunoblot analysis, the glucosyltransferase appeared to be conserved in L. pneumophila strains but was absent from the other Legionella species. This study represents the first identification of a UDP-glucosyltransferase in an intracellular parasite, and therefore modification of a eukaryotic target(s) by this enzyme may influence host cell function and promote L. pneumophila proliferation.