Characterization of putative membrane protein genes of the ‘Candidatus Phytoplasma asteris’, chrysanthemum yellows isolate

2008 ◽  
Vol 54 (5) ◽  
pp. 341-351 ◽  
Author(s):  
Luciana Galetto ◽  
Jacqueline Fletcher ◽  
Domenico Bosco ◽  
Massimo Turina ◽  
Astri Wayadande ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Expression Vector ◽  
Polyclonal Antibodies ◽  
Western Blots ◽  
Protein Fragments ◽  
Aster Yellows ◽  
Euscelidius Variegatus ◽  
Macrosteles Quadripunctulatus ◽  
Aster Yellows Phytoplasma

To characterize potentially important surface-exposed proteins of the phytoplasma causing chrysanthemum yellows (CY), new primers were designed based on the conserved regions of 3 membrane protein genes of the completely sequenced onion yellows and aster yellows witches’ broom phytoplasmas and were used to amplify CY DNA. The CY genes secY, amp, and artI, encoding the protein translocase subunit SecY, the antigenic membrane protein Amp and the arginine transporter ArtI, respectively, were cloned and completely sequenced. Alignment of CY-specific secY sequences with the corresponding genes of other phytoplasmas confirmed the 16S rDNA-based classification, while amp sequences were highly variable within the ‘Candidatus Phytoplasma asteris’. Five CY partial sequences were cloned into the pRSetC expression vector, and 3 of the encoded protein fragments (Amp 64/651, Amp 64/224, ArtI 131/512) were expressed as fusion antigens for the production of CY-specific polyclonal antibodies (A416 against Amp 64/224; A407 against ArtI 131/512). A416 recognized, in Western blots, the full-length Amp from CY-infected plants (periwinkle, daisy) and insect vectors ( Euscelidius variegatus , Macrosteles quadripunctulatus ). A416 also reacted to European aster yellows, to primula yellows phytoplasmas, to northern Italian strains of ‘Ca. Phytoplasma asteris’ from lettuce and gladiolus, but it did not react to American aster yellows phytoplasma.

scholarly journals Further characterization of erythrocyte p80 and the membrane protein defect of In(Lu) Lu(a-b-) erythrocytes

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1475-1481
Author(s):  
MJ Telen ◽  
I Rogers ◽  
M Letarte
Keyword(s):  
Monoclonal Antibody ◽  
Membrane Protein ◽  
Concanavalin A ◽  
Polyclonal Antibodies ◽  
Erythrocyte Ghosts ◽  
Con A ◽  
Down Regulation ◽  
Regulation Of Expression ◽  
Variable Effect

We have previously shown that the In(Lu) gene down-regulates expression of an erythrocyte protein antigen identified by murine monoclonal antibody (MoAb) A3D8. In the present study we have examined In(Lu) Lu(a- b-) erythrocytes for expression of additional epitopes on the erythrocyte 80 kilodalton protein (p80) bearing the A3D8 antigen. Using a total of seven additional MoAbs that recognize three epitopes on erythrocyte p80, we have shown that In(Lu) Lu(a-b-) erythrocytes exhibit down-regulation of expression of all three epitopes. In(Lu) erythrocytes also showed a reduction in their reactivity to rabbit antibodies produced against purified p80 from either erythrocytes or lymphocytes. Furthermore the reactivity of the MoAbs was not altered by treatment of the cells with neuraminidase but was substantially reduced by treatment of cells with trypsin or chymotrypsin. The polyclonal anti- p80 sera were shown to react with a fragment of 50,000 daltons, still associated with erythrocyte ghosts, following treatment of the cells with trypsin or chymotrypsin. Treatment of erythrocytes with the thiol- reactive reagent AET decreased their reactivity with the MoAbs but had a variable effect on their reactivity with polyclonal antibodies. Erythrocyte p80 is a glycoprotein with N-linked oligosaccharides, as demonstrated by its binding to concanavalin A (Con A) and Len culinaris lectins. Following Endoglycosidase F treatment, erythrocyte p80 underwent a reduction in apparent mol wt of 11,000. The presence of a reduced amount of an intact p80 glycoprotein, seen by a decrease in reactivity with MoAbs directed at three distinct epitopes and with two different polyclonal antibodies, suggests that the In(Lu) gene interferes with expression by erythrocytes of the entire p80 glycoprotein.

scholarly journals The Production and characterization of Polyclonal Antibodies Against Interferon Alpha in Mice

2021 ◽  
Vol 3 (4) ◽  
Author(s):  
Mehreen Fatima ◽  
Fatima Khalid ◽  
Azra Quraishi
Keyword(s):  
Immune Response ◽  
Autoimmune Diseases ◽  
Interferon Alpha ◽  
Polyclonal Antibodies ◽  
Mouse Serum ◽  
Interferon Production ◽  
Dot Blot ◽  
Western Blots ◽  
Response Activation

The polyclonal antibodies are used extensively for research purposes in many areas of biology, such as immunoprecipitation, histochemistry, enzyme linked immunosorbent assays (ELISA), diagnosis of disease and western blots. Typically, an animal’s immune system will generate a large group of antibodies that recognize several epitopes of a particular antigen. Interferon alpha plays an important role in immune response activation and therefore is of interest in studies related to autoimmune diseases. In this paper the production of antibodies against interferon was studied in order to quantify interferon production to analyze interferon levels in autoimmune disorders in the future. For the antibody production, one month old laboratory grade mice were injected with interferon alpha in combination with a Freund’s complete adjuvant for a course of five weeks after which the antibodies were obtained in mouse serum. Confirmation of the production of anti-interferon alpha antibodies was carried out by the Elisa, immune dot blot and western blot analysis. An interferon alpha of approximately 20.5-21.5 KDa was detected in immunedot blot test.  These antibodies may be produced in these mouse models commercially and could be used in future for treatment of autoimmune diseases by managing the interferon levels in the patients. Copyright(c) The Authors

Detection and molecular characterization of an aster yellows phytoplasma in parsley

1998 ◽  
Vol 20 (1) ◽  
pp. 55-61 ◽  
Author(s):  
A.-H. Khadhair ◽  
L. M. Kawchuk ◽  
R.C. Taillon ◽  
G. Botar
Keyword(s):  
Molecular Characterization ◽  
Aster Yellows ◽  
Aster Yellows Phytoplasma

EXPRESSION OF THE IMMUNODOMINANT MEMBRANE PROTEIN OF CHLORANTIE-ASTER YELLOWS PHYTOPLASMA IN NICOTIANA BENTHAMIANA FROM A POTATO VIRUS X-BASED VECTOR

2001 ◽  
pp. 409-416 ◽  
Author(s):  
Y. Hong ◽  
D.L. Davies ◽  
R. van Wezel ◽  
B.E. Ellerker ◽  
A. Morton ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Potato Virus ◽  
Nicotiana Benthamiana ◽  
Potato Virus X ◽  
Aster Yellows ◽  
Aster Yellows Phytoplasma

scholarly journals Detection and molecular characterization of phytoplasmas infecting apple trees in Poland

2014 ◽  
Vol 41 (No. 1) ◽  
pp. 27-33 ◽  
Author(s):  
M. Cieślińska ◽  
D.E. Kruczyńska
Keyword(s):  
Rflp Analysis ◽  
Rrna Gene ◽  
Apple Trees ◽  
Aster Yellows ◽  
Candidatus Phytoplasma Mali ◽  
Multiple Alignments ◽  
Pcr Rflp ◽  
Aster Yellows Phytoplasma ◽  
Reference Strains

During 2010&ndash;2012, samples from 225 apple trees growing in six regions of Poland were tested for phytoplasmas. 16S&nbsp;rRNA gene and 16S-23S spacer region sequences were amplified from total DNAs prepared from phloem tissue of apple shoots. According to the results of PCR-RFLP and sequence analyses, apple trees were infected by Candidatus Phytoplasma mali and Ca. P. asteris. Fragments of 16S rDNA plus 16S-23S spacer region of the Ca. P. mali isolates digested with HpaII enzyme showed two restriction profiles: P-I and P-II. Multiple alignments of 16S rRNA gene fragments revealed that the isolates of Ca. P. mali shared 100% sequence identity among themselves as well as with reference strains AT and AP-15 of apple proliferation phytoplasma. The nucleotide sequence of the same region of <br /> Ca. P. asteris isolates confirmed the phylogenetic relationship with reference strains OAY (MIAY) and AY1 of aster yellows phytoplasma PCR-RFLP analysis of ribosomal protein (rpl22 and rpS3), secY, and tuf genes did not show the sequence diversity of the isolates of aster yellows phytoplasma. &nbsp; &nbsp;

scholarly journals Molecular Characterization of Annexin B2, B3 and B12 in Taenia multiceps

Genes ◽  
2018 ◽  
Vol 9 (11) ◽  
pp. 559 ◽  
Author(s):  
Cheng Guo ◽  
Yue Xie ◽  
Yuchen Liu ◽  
Ning Wang ◽  
Jiafei Zhan ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Polyclonal Antibodies ◽  
Parasite Development ◽  
Economic Losses ◽  
Mrna Levels ◽  
Western Blots ◽  
Taenia Multiceps ◽  
Homologous Genes ◽  
Coenurus Cerebralis

Coenurus cerebralis, the metacestode of Taenia multiceps, causes coenurosis, a disease severely affecting goat, sheep, cattle and yak farming and resulting in huge economic losses annually. Annexins bind calcium ions and play an important role in flatworm parasite development. To explore potential functions of annexins in T. multiceps, three homologous genes, namely, TmAnxB2, TmAnxB3 and TmAnxB12, were screened from the transcriptome dataset, amplified from C. cerebralis cDNA and subjected to bioinformatics analysis. Then, polyclonal antibodies recognizing the recombinant TmAnxB2 (rTmAnxB2) and rTmAnxB3 were prepared for localization of TmAnxB2 and TmAnxB3 in different tissues and developmental stages by immunofluorescence. The transcription of all three genes was also measured by relative fluorescent quantitative PCR. The sizes of rTmAnxB2, rTmAnxB3 and rTmAnxB12 were 58.00, 53.06 and 53.51 kDa, respectively, and rTmAnxB12 was unstable. Both rTmAnxB2 and rTmAnxB3 were recognized by goat-positive T. multiceps sera in Western blots. Immunofluorescence revealed that TmAnxB2 and TmAnxB3 were localized in the protoscolex and cyst wall and TmAnxB3 was also detected in adult cortex. TmAnxB2 and TmAnxB12 mRNA levels were determined to be highest in oncospheres and protoscolex, whereas transcription of TmAnxB3 was highest in scolex and immature segments. Taken together, these findings indicate that TmAnxB2 and TmAnxB12 may play critical roles in T. multiceps larvae, while TmAnxB3 may have important functions in adults. These results will lay the foundation for functional research of annexins in T. multiceps.

Molecular Characterization of Aster Yellows Phytoplasma Associated with Valerian and Sowthistle Plants by PCR-RFLP Analyses

2008 ◽  
Vol 156 (6) ◽  
pp. 326-331 ◽  
Author(s):  
A.-H. Khadhair ◽  
C. Hiruki ◽  
M. Deyholos
Keyword(s):  
Molecular Characterization ◽  
Aster Yellows ◽  
Pcr Rflp ◽  
Aster Yellows Phytoplasma

Molecular characterization of mitofilin (HMP), a mitochondria-associated protein with predicted coiled coil and intermembrane space targeting domains

1996 ◽  
Vol 109 (9) ◽  
pp. 2253-2264 ◽  
Author(s):  
P.R. Odgren ◽  
G. Toukatly ◽  
P.L. Bangs ◽  
R. Gilmore ◽  
E.G. Fey
Keyword(s):  
Polyclonal Antibodies ◽  
Limited Proteolysis ◽  
Coiled Coil ◽  
Cell Types ◽  
Cdna Clones ◽  
Intermembrane Space ◽  
Western Blots ◽  
Double Label ◽  
Helical Region

We have identified and characterized a human protein of the mitochondria which we call mitofilin. Using monoclonal and polyclonal antibodies, we have isolated cDNA clones and characterized mitofilin biochemically. It appears as a 90 and 91 kDa doublet in western blots and is translated from a single 2.7 kb mRNA. Antibodies raised against cellular and bacterially-expressed protein given identical cytoplasmic immunofluorescence and immunoblot results. Mitofilin co-localizes with mitochondria in immunofluorescence experiments and co-purifies with mitochondria. Double label studies show co-localization only with mitochondria and not with Golgi or endoplasmic reticulum. Co-localization with mitochondria is retained when actin or tubulin are de-polymerized, and mitofilin is expressed in all human cell types tested. The cDNA encodes a polypeptide with a central alpha-helical region with predicted coiled coil domains flanked by globular amino and carboxy termini. Unlike coiled coil motor proteins, mitofilin is resistant to detergent extraction. The presence of mitochondrial targeting and stop-transfer sequences, along with the accessibility of mitofilin to limited proteolysis suggests that it resides predominantly in the intermembrane space, consistent with immuno-electron micrographs which show mitofilin mainly at the mitochondrial periphery. The cDNA sequence of mitofilin is identical to that recently reported by Icho et al. (1994; Gene 144, 301–306) for a mRNA preferentially expressed in heart muscle (HMP), consistent with the high levels of mitochondria in cardiac myocytes.

Detection and molecular characterization of an aster yellows phytoplasma in rhodiola in Alberta, Canada

2009 ◽  
Vol 116 (4) ◽  
pp. 145-148
Author(s):  
S.-F. Hwang ◽  
J. Feng ◽  
R. Hwang ◽  
S. E. Strelkov ◽  
K. Ampong-Nyarko ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Aster Yellows ◽  
Aster Yellows Phytoplasma
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