The molecular basis for genetic polymorphism of human deoxyribonuclease I: identification of the nucleotide substitution that generates the fourth allele

AbstractIn addition to the three alleles commonly responsible for the protein polymorphism of human deoxyribonuclease I, a mutation encoded by a fourth allele, DNASEI*4, was detected by isoelectric focusing. All 8 exons covering the entire open reading frame of the human DNase I gene were amplified by the polymerase chain reaction and subjected to direct sequencing. Only one nucleotide substitution, a C-to-G transition (CAG → GAG), in the codon for amino acid 9 of the mature enzyme was found. This substitution resulted in the replacement of Gln with Glu (Q9E)

Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci

A glycosylation island is a genetic region required for glycosylation. The glycosylation island of flagellin in Pseudomonas syringae pv. tabaci 6605 consists of three orfs: orf1, orf2 and orf3. Orf1 and orf2 encode putative glycosyltransferases, and their deletion mutants, Delta orf1 and Delta orf2, exhibit deficient flagellin glycosylation or produce partially glycosylated flagellin respectively. Digestion of glycosylated flagellin from wild-type bacteria and non-glycosylated flagellin from Delta orf1 mutant using aspartic N-peptidase and subsequent HPLC analysis revealed candidate glycosylated amino acids. By generation of site-directed Ser/Ala-substituted mutants, all glycosylated amino acid residues were identified at positions 143, 164, 176, 183, 193 and 201. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis revealed that each glycan was about 540 Da. While all glycosylation-defective mutants retained swimming ability, swarming ability was reduced in the Delta orf1, Delta orf2 and Ser/Ala-substituted mutants. All glycosylation mutants were also found to be impaired in the ability to adhere to a polystyrene surface and in the ability to cause disease in tobacco. Based on the predicted tertiary structure of flagellin, S176 and S183 are expected to be located on most external surface of the flagellum. Thus the effect of Ala-substitution of these serines is stronger than that of other serines. These results suggest that glycosylation of flagellin in P. syringae pv. tabaci 6605 is required for bacterial virulence. It is also possible that glycosylation of flagellin may mask elicitor function of flagellin molecule

Identification of DNA methylated regions by using methylated DNA immunoprecipitation sequencing in Brassica rapa

DNA methylation is an epigenetic gene regulatory mechanism that plays an essential role in gene expression, transposon silencing, genome imprinting and plant development. We investigated the influence of DNA methylation on gene expression in Brassica rapa L., to understand whether epigenetic differences exist between inbred lines. Genome-wide DNA methylation was analysed by methylated DNA immunoprecipitation sequencing (MeDIP-seq) of 14-day-old first and second leaves from two inbred lines of Chinese cabbage, one susceptible and one resistant to fusarium yellows caused by Fusarium oxysporum f. sp. conglutinans. MACS (model-based analysis for ChIP-seq) identified DNA methylation peaks in genic regions including 2 kb upstream, exon, intron and 2 kb downstream. More than 65% of genes showed similar patterns of DNA methylation in the genic regions in the two inbred lines. DNA methylation states of the two inbred lines were compared with their transcriptome. Genes having DNA methylation in the intron and in the 200 bp upstream and downstream regions were associated with a lower expression level in both lines. A small number of genes showed a negative correlation between differences in DNA methylation levels and differences in transcriptional levels in the two inbred lines, suggesting that DNA methylation in these genes results in transcriptional suppression

The role of FRIGIDA and FLOWERING LOCUS C genes in flowering time of Brassica rapa leafy vegetables

© 2019, The Author(s). There is a wide variation of flowering time among lines of Brassica rapa L. Most B. rapa leafy (Chinese cabbage etc.) or root (turnip) vegetables require prolonged cold exposure for flowering, known as vernalization. Premature bolting caused by low temperature leads to a reduction in the yield/quality of these B. rapa vegetables. Therefore, high bolting resistance is an important breeding trait, and understanding the molecular mechanism of vernalization is necessary to achieve this goal. In this study, we demonstrated that BrFRIb functions as an activator of BrFLC in B. rapa. We showed a positive correlation between the steady state expression levels of the sum of the BrFLC paralogs and the days to flowering after four weeks of cold treatment, suggesting that this is an indicator of the vernalization requirement. We indicate that BrFLCs are repressed by the accumulation of H3K27me3 and that the spreading of H3K27me3 promotes stable FLC repression. However, there was no clear relationship between the level of H3K27me3 in the BrFLC and the vernalization requirement. We also showed that if there was a high vernalization requirement, the rate of repression of BrFLC1 expression following prolonged cold treatments was lower

Generation of Human Antigen-Specific Monoclonal IgM Antibodies Using Vaccinated “Human Immune System” Mice

Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the 'humanization' of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique. After transplantation with CD34+CD38⁻ human hematopoietic progenitor cells, BALB/c Rag2⁻/⁻IL-2Rγc⁻/⁻ mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. "Human Immune System" mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19+CD27+ B cells were retrovirally transduced with the human B cell lymphoma (BCL)-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively. This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigen

Nonabelian Discrete Family Symmetry to Soften the SUSY Flavor Problem and to Suppress Proton Decay

Family symmetry could explain large mixing of the atmospheric neutrinos. The same symmetry could explain why the flavor changing current processes in supersymmetric standard models can be so suppressed. It also may be able to explain why the proton is so stable. We investigate these questions in a supersymmetric, renormalizable extension of the standard model, which possess a family symmetry based on a binary dihedral group Q_6. We find that the amplitude for \mu \to e+\gamma enjoys a suppression factor proportional to |(V_{MNS})_{e3}| ~ m_e/(\sqrt{2}m_\mu) ~ 3.4\times 10^{-3}, and that B(p \to K^0 \mu^+)/B(p \to K^0 e^+) ~ |(V_{MNS})_{e3}|^2 ~ 10^{-5}, where V_{MNS} is the neutrino mixing matrix.Comment: 35 pages, 26 figure

Development and Function of Invariant Natural Killer T Cells Producing TH2- and TH17-Cytokines

Four distinct subsets of invariant natural killer T (NKT) cells are shown to differentiate in the thymus, then migrate to peripheral tissues where they retain their phenotypic and functional characteristics

Detection of COL1A1-PDGFB Fusion Transcripts in Dermatofibrosarcoma Protuberans

Background: Dermatofibrosarcoma protuberans (DFSP) is an uncommon infiltrative tumor of the dermis and subcutaneous tissue. Recent cytogenetic studies have demonstrated a chromosomal translocation of the collagen type I alpha 1 (COL1A1)gene on chromosome 17 to the platelet-derived growth factor B-chain (PDGFB) gene on chromosome 22. Various exons of the COL1A1 gene have been reported to be involved in the fusion with exon 2 of the PDGFB gene. M ethod: The COL1A1-PDGFB fusion transcript was detected by reverse transcriptase-polymerase chain reaction (RT-PCR)using frozen tissue from 4 DFSP patients. Nucleotide sequence analyses were carried out using the PCR products to identify the breakpoints. Results : COL1A1-PDGFB fusion transcripts were detected in all tumor specimens. Sequence analyses revealed that the end of exon 25, 45, 32, or 11 in the COL1A1 gene was fused with the start of exon 2 in the PDGFB gene. Conclusion : Detection of this aberrant fusion transcript can be useful as a diagnostic method for DFSP

Expression of actin genes in the arrow worm Paraspadella gotoi (Chaetognatha)

Arrow worms (the phylum Chaetognatha), one of the major marine planktonic animals, exhibit features characteristic to both deuterostomes and protostomes, and their ancestry therefore remains unknown. As the first step to elucidate the molecular bases of arrow worm phylogeny, physiology and embryology, we isolated cDNA clones for three different actin genes (PgAct1, PgAct2 and PgAct3) from the benthic species Paraspadella gotoi, and examined their expression patterns in adults and juveniles. The amino acid sequences of the three actins resembled each other, with identities ranging from 86% to 92%. However, the patterns of the spatial expression of the genes were independent. The PgAct1 gene might encode a cytoplasmic actin and was expressed in oogenic cells, spermatogenic calls, and cells in the ventral ganglion. The PgAct2 and PgAct3 genes encoded actins of divergent types. The former was expressed in well-developed muscle of the head (gnathic) region and trunk muscle cells, whereas the latter was expressed in muscle of the trunk and tail regions and oogenic cells. These results suggest that, similarly to other metazoans, the chaetognath contains multiple forms of actins, which are expressed in various manners in the adult and juvenile arrow worm