531 Matching Annotations
  1. Last 7 days
    1. ZFIN: ZDB-ALT-051111-6

      DOI: 10.1016/j.devcel.2020.07.015

      Resource: (ZFIN Cat# ZDB-ALT-051111-6,RRID:ZFIN_ZDB-ALT-051111-6)

      Curator: @Naa003

      SciCrunch record: RRID:ZFIN_ZDB-ALT-051111-6


      What is this?

    2. ZFIN: ZDB-ALT-051223-6

      DOI: 10.1016/j.devcel.2020.07.015

      Resource: (ZFIN Cat# ZDB-ALT-051223-6,RRID:ZFIN_ZDB-ALT-051223-6)

      Curator: @Naa003

      SciCrunch record: RRID:ZFIN_ZDB-ALT-051223-6


      What is this?

    3. ZFIN: ZDB-GENO-200316-6

      DOI: 10.1016/j.devcel.2020.07.015

      Resource: (ZFIN Cat# ZDB-GENO-200316-6,RRID:ZFIN_ZDB-GENO-200316-6)

      Curator: @Naa003

      SciCrunch record: RRID:ZFIN_ZDB-GENO-200316-6


      What is this?

  2. Mar 2021
    1. CONSEQUENCES JURIDIQUES DU DEFAUT D’ACCUSE DE RECEPTION. Article L112-6 CRPASi l’administration n’accuse pas réception de la demande, ou le fait demanière incorrecte (mentions incomplètes ou erronées), les délais derecours ne sont pas opposables à l’auteur de la demande.L’usager pourra donc contester votre décision implicite à tout momentdevant la juridiction administrative.Il est donc important pour la sécurité juridique de vos décisions, derespecter les règles tant sur la forme que sur le fond.
    1. Results for individual PALB2 variants were normalized relative to WT-PALB2 and the p.Tyr551ter (p.Y551X) truncating variant on a 1:5 scale with the fold change in GFP-positive cells for WT set at 5.0 and fold change GFP-positive cells for p.Y551X set at 1.0. The p.L24S (c.71T>C), p.L35P (c.104T>C), p.I944N (c.2831T>A), and p.L1070P (c.3209T>C) variants and all protein-truncating frame-shift and deletion variants tested were deficient in HDR activity, with normalized fold change <2.0 (approximately 40% activity) (Fig. 1a).

      AssayResult: 4.7

      AssayResultAssertion: Normal

      StandardErrorMean: 0.32

    2. A total of 84 PALB2 patient-derived missense variants reported in ClinVar, COSMIC, and the PALB2 LOVD database were selected

      HGVS: NM_024675.3:c.109C>T p.(Arg37Cys)

    1. SUPPLEMENTARY DATA

      AssayResult: 93.06

      AssayResultAssertion: Not reported

      PValue: > 0.9999

      Comment: Exact values reported in Table S3.

    2. To this end, 44 missense variants found in breast cancer patients were identified in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar) and/or selected by literature curation based on their frequency of description or amino acid substitution position in the protein (Supplemental Table S1).

      HGVS: NM_024675.3:c.226A>G p.(Ile76Val)

    1. Source Data

      AssayResult: 17.43

      AssayResultAssertion: Abnormal

      ReplicateCount: 2

      StandardErrorMean: 5.19

      Comment: Exact values reported in “Source Data” file.

    2. Source Data

      AssayResult: 7.82

      AssayResultAssertion: Abnormal

      ReplicateCount: 2

      StandardDeviation: 2.31

      StandardErrorMean: 1.64

      Comment: Exact values reported in “Source Data” file.

    3. We, therefore, analyzed the effect of 48 PALB2 VUS (Fig. 2a, blue) and one synthetic missense variant (p.A1025R) (Fig. 2a, purple)29 on PALB2 function in HR.

      HGVS: NM_024675.3:c.1227_1231delTGTTA p.(Y409X)

    1. Most Suspected Brugada Syndrome Variants Had (Partial) Loss of Function

      AssayResult: 0

      AssayResultAssertion: Abnormal

      ReplicateCount: 39

      StandardErrorMean: 0

      Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

    2. we selected 73 previously unstudied variants: 63 suspected Brugada syndrome variants and 10 suspected benign variants

      HGVS: NM_198056.2:c.1100G>T p.(Arg367Leu)

  3. Feb 2021
    1. RRID:ZFIN_ZDB-ALT-160322-6

      DOI: 10.1111/bph.15156

      Resource: (ZFIN Cat# ZDB-ALT-160322-6,RRID:ZFIN_ZDB-ALT-160322-6)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-ALT-160322-6


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    1. RRID:ZDB-ALT-040217-6

      DOI: 10.7554/eLife.37001

      Resource: (ZFIN Cat# ZDB-ALT-040217-6,RRID:ZFIN_ZDB-ALT-040217-6)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-ALT-040217-6


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    2. RRID:ZDB-ALT-110310-6

      DOI: 10.7554/eLife.37001

      Resource: (ZFIN Cat# ZDB-ALT-110310-6,RRID:ZFIN_ZDB-ALT-110310-6)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-ALT-110310-6


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    1. RRID:ZFIN_ZDB-ALT-051223-6

      DOI: 10.7554/eLife.42881

      Resource: (ZFIN Cat# ZDB-ALT-051223-6,RRID:ZFIN_ZDB-ALT-051223-6)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-ALT-051223-6


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    2. RRID:ZFIN_ZDB-ALT-160323-6

      DOI: 10.7554/eLife.42881

      Resource: (ZFIN Cat# ZDB-ALT-160323-6,RRID:ZFIN_ZDB-ALT-160323-6)

      Curator: @scibot

      SciCrunch record: RRID:ZFIN_ZDB-ALT-160323-6


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    1. Supplemental material

      AssayResult: 89, 90

      AssayResultAssertion: Normal

      Comment: See Table S3 for details

    2. Supplemental material

      AssayResult: 5.8, 6.1

      AssayResultAssertion: Abnormal

      Comment: See Table S3 for details

    3. We analysed a total of 82 blood samples derived from 77 individuals (online supplemental table 3). These 77 individuals corresponded either to new index cases suspected to harbour a pathogenic TP53 variant or to relatives of index cases harbouring TP53 variants.

      HGVS: NM_000546.5:c.1043T>C p.(Leu348Ser)

  4. Jan 2021
    1. PTT

      AQ:6 Please provide an expansion for PTT.

      PTT: partial thromboplastin time

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  5. Oct 2020
    1. He who sees me everywhereand sees everything in mewill not be lost to me,and I will not be lost to him

      Is it explaining the relationship between lord Krishna and Arjuna?

    2. A man of discipline should alwaysdiscipline himself, remain in seclusion,isolated, his thought and self well controlled,without possessions or hope.

      A man of discipline is the one who should control his senses, and he should be discipline by himself.

    1. I am very intrigued by the arguments that are mentioned by Angeline Grimke'. These are perspectives that I have not considered other than the morality aspect situation. I do like the way that she compares America, where we enslave men, women, and children but even in Africa they would be free. Not only free but have many more inalienable rights in such a poor, egregious country, in my opinion of the times anyway. But here in the land of the free they can't control one single facet of their life. And how exactly does a person being born in America, that is as American as George Washington, lose his rights because one man says so and believes that he is subservient in nature. I would not have done well back then. The ignorance that they posses is devastatingly incredible. That total lack of consideration for other people and human life is utterly despicable. To think that the men could not even get it figured out. It was such a long lasting debate that women had to get involved and try to speak some sort of since to these so called men. I know it is a different time but I am unable to fathom treating someone so horribly. I am glad it is a different time!

  6. Sep 2020
    1. The answer is that they were two different “Yahweh’s.”

      OK. I'm trying to work with this. I feel like I've been on the edge of revelation concerning this for quite a while.

      • Wouldn't the 1st encounter w/ a negative entity be in Genesis (Specifically because of all the killing and wars and Gen 6 account)
      • Was the serpent a negative entity????
    1. She persuades Anu, her father, to give her the fiery Bull of Heaven (the constellation Taurus) so that she can punish Gilgamesh with death.

      Why she wanted to kill Gilgamesh, if she loves him?

  7. Aug 2020
    1. Los Acuerdos PDET, así sean aún incipientes y en construcción, concebidos como procesos, deberían dar lugar a proyectos y acciones concretos, en algunos casos de tipo experimental o piloto. Esto, por ejemplo, en cuanto a proyectos productivos de la estrategia de “territorios agroalimentarios

      Se responde en parte la pregunta de cómo se articulan Territorios agroalimentarios campesinos y PDET

  8. Jun 2020
    1. L’article 6 fixe un second principe général qui reconnait le droit inhérent à la vie de tout enfant et l’obligation de l’État d’assurer sa survie et son développement
  9. Jan 2020
    1. RRID:ZFIN_ZDB-ALT-150424-6

      DOI: 10.7554/eLife.42455

      Resource: (ZFIN Cat# ZDB-ALT-150424-6,RRID:ZFIN_ZDB-ALT-150424-6)

      Curator: @evieth

      SciCrunch record: RRID:ZFIN_ZDB-ALT-150424-6


      What is this?

    1. down-slanting eyelid
    2. short proximal extremities
    3. 6
    4. 6
    5. 6
    6. 6
    7. 6
    8. macrocephaly
    9. muscular hypotonia
    10. intellectual disability
    11. motor development
    12. speech and language development
    13. developmental delay
  10. Dec 2019
  11. Sep 2019
  12. Jul 2019
    1. 5. To accept the collaboration in Serbia of representatives of the Austro-Hungarian Government in the suppression of the subversive movement directed against the territorial integrity of the monarchy;

      This demand was not met leading to be one of the causes of WWI.

    1. The removal, so far as possible, of all economic barriers and the establishment of an equality of trade conditions among all the nations consenting to the peace and associating themselves for its maintenance.

      Free trade

    2. Adequate guarantees given and taken that national armaments will be reduced to the lowest point consistent with domestic safety.

      reduction of national weapons

    1. Prevalence of type III secretion system genes amongV. parahaemolyticusfrom Cochin estuary, shrimp farm and seafood
    2. Production of chitinase
    1. Resistance to ampicillin
    2. Citrate utilisation tes
    3. Nitrate reduction test
    4. Production of β-galactosidase (ortho-Nitrophenyl β-D-galactopyranoside (ONPG)) test
    5. Urease production
    6. Gelatinase production
    7. Growth at different temperatures
    8. Salt tolerance tes
    9. Voges Proskauer (acetoin production) test
    10. Indole production
    11. Carbon source utilisation tes
    12. Carbohydrate fermentation test
    13. Amino acids utilisation test (Decarboxylase/dihydrol
    14. Species level identification
    15. Urease productio
    16. Gelatinase productio
    17. Growth at different temperature
    18. Salt tolerance tes
    19. Voges Proskauer (acetoin production) tes
    20. Indole production
    21. Carbon source utilisation t
    22. Carbohydrate fermentation tes
    23. Amino acids utilisation test (Decarboxylase/dihydrola
    24. Species level identification
    1. Relative antibiotic resistance amongVibrioisolated from Cochin estuary, shrimp farm and seafood
  13. Jun 2019
    1. Dominance of regulatory transitional B cellsand lower memory B cells in HBsAgpositive compared to negative newborns
    1. V8 protease-mediated semisynthesis of a globin was carried out at 4°C in 0.05 M ammonium acetate buffer (pH 6) containing 30% 1-propanol. For this, the lyophilized samples of natural or synthetic analogs of a 1-30 and respective a31-141 were individually prepared in water. Suitable volumes of the complementary fragments were mixed to obtain a 1:1 molar ratio and lyophilized. The lyophilized material was dissolved in appropriate amount of ammonium acetate buffer (pH 6). To this solution, a suitable volume of 1-propanol was added to a final concentration of 30% 1-propanol and 20 mg/ml substrate. The mixture was cooled on ice subsequent to which suitable volume of V8 protease solution prepared in water ( 1% w/w of substrate) was added. The ligation reaction mixture was incubated at 4°C for 24 hours. The extent of synthesis was monitored on RPHPLC by loading an aliquot of the reaction mixture on an analytical reverse phase column. The reaction was stopped by addition of chilled 5% TF A solution (0.2 fold v/v) and lyophilized
    2. Construction of mutant a globins
  14. May 2019
    1. The reaction mixture contained 0.2 mL of enzyme sample, 0.3 mL of buffer and 0.5 mL of p-nitrophenyl-β-D-glucopyranoside (1.0 mM) prepared in 100 mM buffer as the substrate. The reaction was terminated after 30 min of incubation at 70 °C by adding 2 mL of sodium carbonate-bicarbonate buffer (0.1 M, pH 10.0). The liberation of p-nitrophenol was measured at 400 nm and its yield was determined using a standard curve of p-nitrophenol (1-10 μg mL-1) prepared in sodium carbonate-bicarbonate buffer
    2. β-Glucosidase
    3. Overnight grown cultures of E. coli DH5α, E. coli BL21 (DE3), E. coli XL1blue cells with and without constructs were preserved in 80 % v/v glycerol
    4. MAINTENANCE OF THE RECOMBINANT STRAIN
    1. incubated on ice for 5 min, buffer N3 (350 J.!l) was added to the mixture and the tube was iriverted 4-6 times until mix appeared cloudy. Cell debris was removed by centrifugation at 12000 x g for 10 min and the supernatant was applied to QIAprep spin columns. Columns were centrifuged at 12000 x g for 1 miri and the flow through was discarded and columns were washed using 750 J.!l of 70% ethanol and centrifuged, at 12000 x g for 1 min. Additional centrifugation was performed to remove the residual ethanol. The columns were placed in a 1.5 ml microfuge tube and DNA was eluted with autoclaved water or 1 mM Tris-HCI (PH 8.0).
    2. Plasmid DNA was extracted using commercially available kit (Qiagen, Germany) as per manufacturer's instructions. For a miniprep, bacterial cell pellet from 5ml freshly grown culture were resuspended in 250 III buffer PI containing RNaseA in a microfuge tube, followed by lysis in 250 III of buffer P2. After the tube was
    3. Plasmid DNA Isolation
    4. medium. The culture volume in 75 cm2 culture flasks, was increased to 25 ml from 12 ml. The flasks were kept at 37°C and the medium was prewarmed before use. The flasks were gassed with a mixture of 5% C02, 3% 02 and 92% N2 for a i minimum of 20 seconds at a pressure of around 5 Ib/in2. The culture medium was changed daily without the addition of RBCs. Blood smears were prepared once or twice a week to check the ~tate of the cultures and the presence of gametocytes. Typically, mature gametocytes were observed after 14-17 days
    5. Fresh stock of parasites was thawed for culture as described above. Thin blood smears were made on the fourth day after setting up the culture. When high , parasitemia with "stressed" parasites was observed, culture volume was increased by the addition of medium. At this stage, fresh RBCs were not added to the culture
    6. Gametocyte cultures
    1. 700 mM NaCl, 12.5 mM CaCh, pH 7.4). 5 J.!L of Annexin-V conjugated to Alexa fluor 488 and 1 J.!L of working solution of PI (100 Jlg/mL) were added to the 100 J.!L cell suspension. Cells were incubated for 15 min at room temperature. Following this, 400 J.!L of IX Annexin binding buffer was added to dilute the sample. The samples were placed on ice. The fluorescence was measured by flow cytometry in FL 1 and FL2 channels for Annexin-V-Alexa fluor 488 and PI fluorescence respectively.
    2. The Vybrant apoptosis assay kit was used to perform Annexin-V/PI staining as described previously (3). The assay is based on the principle that apoptotic cells show loss of membrane asymmetry by exposing phosphatidylserine on the outer surface of the plasma membrane for which Annexin-V, a phosphlipid binding protein, shows high affinity. Hence, Annexin-V conjugated to Alexa fluor 488 binds to phosphatidylserine exposed on apoptotic cells, while propidium iodide binds to nucleic acids of all non-viable cells including necrotic and apoptotic cells. Thus, flow-cytometric analysis of Annexin-V /PI stained cells reveals distinct cellular populations, with the viable cells displaying little or no fluorescence; the early apoptotic cells show green fluorescence of Annexin-V conjugated to Alexa fluor 488; the late apoptotic cells display both green and red fluorescence, while necrotic cells show red fluorescence. The cells after appropriate treatment were harvested by centrifugation at 250 x g for 5 min and were given two washes with ice-cold 1X PBS following which they were resuspended in 100 J!L of ice-cold 1X Annexin binding buffer (50 mM HEPES,
    3. Assay for detection of apoptosis by Annexin-V /PI staining
    1. function of resolution. The displacement for an isotropic B-value is related to the displacement u by the equation B = 8;(u2). The isotropic B-value assumes equal movements in all the directions. However, the vibration of an atom need not be the same in all the directions, and in such a case motion is described by anisotropic displacement parameter. In this formulation the motion is described by an ellipsoid that can be rotated in any direction. The entire anisotropic displacement can be described in terms of six elements: UIJ, U22 and U33 specify the magnitude of movement in three axis and U 12, U t3 and U23 specify the rotation off the principal axis. Anisotropic displacement parameters can be converted to the isotropic equivalent by the formula Biso = 8;(Ull+l'22+U33). The B-values are restrained during refinement. Atoms that are bonded to each other influence each other's motion. B-values are restrained in such a manner that the average difference in the B-values of bonded atoms is kept to a target value. The B-values should vary smoothly along the protein chain and within the side chain. The usual target restraint for adjacent bonded main chain atom is 1.0 and for side chain the target value is 1.5 since one end of the side chain is free, ensuring the higher gradient. Similarly B-values can be graded for the one to three members of a bond angle. For main chain angles the target value is 1.5 and for the side chain angle the value is set to 2.0. Like rigid body refinement, it is done at the early stages of the refinement process. Refinement of the atomic B-factors is a bit tricky and is carried out in later stages of refinement.
    2. The individual atoms were then refined by several cycles of conventional positional refinement, which uses the conjugate gradient minimization method. The proper weight term called Wa was calculated which was used for subsequent positional refinement (Brunger et al., 1987). In case of CNS this value was set to -1 and the program itself calculated these weights. The refinement was started using data in the range 50 A -4.0 A and higher resolution data were added in a stepwise fashion. After complete data had been added, the F0-Fc and 2F0-Fc electron density maps in addition to the composite omit map were calculated and displayed on an HP xw8400workstation (Hewlett-Packard Company, U.S.A.) using Coot (Emsley P, 2004 ). The electron density map was examined in the context of the model and the regions of the map where the electron density was not satisfactory or the model did not fit the density were identified. Using Coot, the residues were mutated to the sequence of the molecule of interest and wherever required, moved locally to correspond to the visible density. This was followed by refinement to check if the changes made could be accepted or not. Depending on the resolution to which X-ray data was available, anisotropic or individual B-factors were also refined. This process of model building and refinement was carried out iteratively until all the differences in sequence with the probe model had been accounted for and there was no ambiguity in the fit between the model and the electron density. Water molecules were included in the model using the 'water pick' program available in CNS only after a sufficient level of refinement had been achieved. This was followed by visual examination of the waters to avoid inclusion of spurious water molecules. The B-value is the measurement of the displacement of an atom from thermal motion, conformational disorder and static lattice disorder. This vibration will smear out the electron density and will also decrease the scattering power of the atom as a
    3. sum runs over all the reflections in your data set, and k is a scale factor needed to put the Fe on the same scale as the Fobs· A model consists typically of five parameters for each atom: x,y,z, B, and Q. The triplet (x,y,z) specifies the position of each atom in an orthogonal coordinate system. B is the B-factor or temperature factor of each atom, and it is related to the thermal motion of the atom. B-factor also contains information about other types of "disorder" including errors that you are being made while constructing and refining model. Q is the occupancy and it is the fraction of time that the atom spends at position (x,y,z). Typically, Q=l. If one has data better than about 1.8 A, then occupancies between zero and one are sometimes used. Refinement procedures for antibodies involve two basic procedures, rigid body refinement followed by positional refinement. Rigid body refinement is used to refine the results obtained from MR in terms of orientation and position of the starting model in the unit cell. Positional refinement is used to refine the positions of individual atoms in space. Both conventional R-factor (Rcryst) and the free R-value (Rfree) (BrUnger, 1992) were used to monitor the progress of refinement. 10% of the reflections were set aside at random to monitor the Rfree during refinement. Rigid body refinement was carried out to further refine the positioning of the probe molecule in the target unit cell. The probe models that gave the highest correlation coefficients were thus subjected to rigid body refinement. Refinement was initially done using data in the range of 50 A -4 A; thereafter data up to the maximum available resolution were added in a step wise manner. The Fab molecule can be defined as an assembly of four domains, the VH, VL, CH and CL. Consequently, rigid body refinement where these domains were considered as discrete rigid units was carried out.
    4. This section describes the detailed refinement strategies used for structure determination. The structures presented in the thesis were refined using CNS (Brunger, 2007; Brunger et al., 1998). The refinement of structures obtained after molecular replacement was done using Crystallography and NMR system (CNS) suite of programs (BrUnger et al., 1998) based on the refinement of crystal structures using Cartesian (BrUnger et al., 1987) or torsion-angle molecular dynamics (Rice and BrUnger, 1994). This task file automatically computes a cross-validated crA estimate, determines a weighting scheme between the X-ray refinement target function and the geometric energy function, refines a flat bulk solvent model (Jiang and Brunger, 1994) and an overall anisotropic B-value of the model by least squares minimization, and subsequently refines the atomic positions. Available target functions include the maximum-likelihood functions MLF, MLI and MLHL (Pannu et al., 1998). Refinement is an iterative process in which the atomic model is modified, structure factor amplitudes are calculated from the modified model, and the agreement between these calculated structure factor amplitudes (IFcl) and the experimental or observed ones (IF ~bsD is determined. The goal is to find the model that produces the best agreement between lfohsl and lfcl. Refinement is a problem of finding the minimum of a function that mathematically expresses the agreement between IFobsl and lfcl. This function is called a target function. A commonly used target function is the crystallographic residual: SUM {(IFobsl -k1Fcl)2}, where the
    5. Structure refinement using the Crystallography and NMR system (CNS) suite
    1. A control reaction was done with the control annealed DNA lacking the oligonucleotide. The constituents were mixed and incubated on ice for 2 min. afterwards, at room temperature, for 5 min. The reaction was further carried out at 37 °C for 2 h followed by heating at 70 °C for 10 min. 12.5 J.tl of the reaction products were analyzed on an agarose gel along with annealed samples to check the complementary strand synthesis. The samples were diluted I 0-times with water and 5 J.ll of the diluted sample was used to transform 50 J.ll of E. coli host strain, DH5a cells. The suspension was pl~!ed on 2 LB agar plates containing ampicillin. Single colonies were picked, grown in liquid culture and miniprep screening of DNA was done to select the positive clones.
    2. Annealed DNA 10.0 J.ll I OX synthesis buffer 2.5 J.ll 1 OX ligation buffer 1.0 J.ll dNTP mix (100 mM) 1.0 J.ll ATP (100 mM) 0.25 J.ll ~ T7 DNA polymerase (5 U/J.tl) 0.5 J.ll T4 DNA ligase (400 U/J.tl) 0.5 J.ll H20 9.25 J.ll Total 25.0 J.l
    3. Titration of uracil containing template: The crude preparation of the phagemid DNA (1 ml aliquot), was used to titrate the uridine incorporation in the template. The strains CJ236 (ung-duf) and DH5aF'(ung+ dut+) of E. coli were transformed with the diluted template DNA The CJ236 cells, plated in the presence of ampicillin and chloramphenicol while DH5aF' cells, plated in the presence of ampicillin alone, were grown overnight on LB agar plates. The good incorporation of uridine gave no colonies or very few colonies in DH5aF' cells whereas with CJ236 several colonies were obtained. A ratio of 103-104 between the number of colonies in CJ236 to that in DH5aF' cells was considered ideal for an efficient incorporation of uridine. Phosphorylation of the mutagenic oligonucleotide: The components of a standard reaction to carry out the phosphorylation are described below Oligonucleotide (180 nmoVml) 1.0 J.ll 1 OX Kinase buffer 2.5 J.ll 10mMATP 1.0 J.ll 10 mM spermidine 0.25 J.ll 100mMDTT 1.25 J.ll T4 polynucleotide Kinase 0.5 J.ll H20 18.5 J.ll Total 25.0 J.LI The constituents were . mixed thoroughly, incubated at 37 °C for 30 min. and subsequently, the enzyme was denatured by heat inactivation at 70 °C for 10 min. Annealing of the mutagenic oligonucleotide: 750 ng (approximately) of the uracil containing single stranded template and 1 J.L) of the phosphorylated· oligonucleotide were taken in IX annealing buffer making up the total reaction volume 20 J.LI. A control reaction, was also carried out simultaneously, lacking the oligonucleotide. The contents were mixed by vortexing and incubated at 95 °C for 10 min. in a water bath. The reaction mixtures were further incubated at 80 °C for I 0 min. in a heat block and the heat block was transferred to ambient temperature, cooled slowly to about 30 °C over a period of 30-60 min. Complementary DNA strand synthesis: The oligonucleotide annealed uracil containing template was used for complementary strand synthesis in the following reaction.
    4. Site-directed mutagenesis was done according to the method employed by Kunkel et al., 1987. Preparation of Uracil containing phagemid: E. coli strain CJ236 was transformed with the required template DNA and grown on LB plates containing the antibiotics, ampicillin 100 Jlg/ml and chloramphenicol 30 Jlg/ml (stock solution made in alcohol). Further, the plates were incubated at 37 °C for 12 h and a single colony was picked from the center of the plate, inoculated in 5 ml LB containing ampicillin and chloramphenicol. The liquid culture was grown at 37 °C overnight with vigorous. shaking. About 500 Jll of the culture was diluted 40-times with LB containing ampici11in and chloramphenicol and grown at 37 °C with vigorous shaking (200 rpm) upto an OD600 of 0.25-0.3. The speed of shaker was reduced to 100 rpm and the culture was left for 30 min. for the F pilus to grow. Afterwards, it was infected with VCS Ml3 helper phage at an MOl of 1:20. The cells were grown for 30 min. in a stationary culture to allow the phages to infect, followed by slow shaking ( 100 rpm) for one hour. Subsequently, the culture was diluted 10-times with 2X YT medium containing ampicillin and chloramphenicol and grown in the presence of 0.25 Jlg/ml uridine and 50 Jlg/ml kanamycin at 37 °C overnight with vigorous shaking. The following day, the culture was chilled on ice for 10 min. and centrifuged at 12,000 rpm for 10 min. at 4 °C in a Sorvall RC5C centrifuge using a GSA rotor. The pellet was discarded and the supernatant was centrifuged again in fresh GSA bottles. A small aliquot of about 1 ml from the supernatant was saved for titration and the . precipitation of the single stranded phagemid was carried out using 0.15 volume of 16.67% PEG in 3.3 M NaCl followed by incubation on ice for 4 h. J'he mixture was centrifuged at 12,000 rpm at 4 °C for 30 min. using a GSA rotor and the pellet was resuspended in 3 ml TE buffer. The suspension was centrifuged at 15,000 rpm at 4 °C for 10 min. using a SS34 rotor. The supernatant was ultracentrifugated at 100,000 g at 4 °C for 2.5 h. The pellet was resuspended in 500 Jll TE buffer followed by phenol-chloroform extraction and precipitation of the single stranded DNA with ethanol for 30 min. at -70 °C. The DNA pellet was washed with 70% ethanol, dried and dissolved in 200 Jll TE buffer. The uracil containing template was quantitated by analysing on an agarose gel.
    5. Site-directed Mutagenesis
    1. Human oocytes were washed twice with PBS containing 0.1 % BSA and then incubated with 1 :50 dilution of immune or pre-immune serum samples at RT for 30 min. Following washing with PBS (3 changes of 5 min each), the oocytes were treated with goat anti-rabbit Ig-FITC conjugate for 30 min at RT. After washing with PBS, the treated oocytes were mounted in Glyceroi:PBS (9: 1) and examined under fluorescent microscope.