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Dec 2019

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Estimation of the Minimum Number of Replication Origins Per Chromosome in any Organism
任何生物体内每个染色体的最小复制起源点数目的估计   

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Abstract

Eukaryote nuclear genomes predominantly replicate through multiple replication origins. The number of replication origins activated per chromosome during the S-phase duration may vary according to many factors, but the predominant one is replication stress. Several studies have applied different approaches to estimate the number and map the positions of the replication origins in various organisms. However, without a parameter to restrict the minimum of necessary origins, less sensitive techniques may suggest conflicting results. The estimation of the minimum number of replication origins (MO) per chromosome is an innovative method that allows the establishment of a threshold, which serves as a parameter for genomic approaches that map origins. For this, the MO can be easily obtained through a formula that requires as parameters: chromosome size, S-phase duration, and replication rate. The chromosome size for any organism can be acquired in genomic databanks (such as NCBI), the S-phase duration can be estimated by monitoring DNA replication, and the replication rate is obtained through the DNA combing approach. The estimation of MO is a simple, quick, and easy method that provides a new methodological framework to assist studies of mapping replication origins in any organism.

Keywords: DNA replication (DNA复制), Replication origins (复制起源点), Replication rate (复制速度), S-phase duration (S期持续时间), Chromosome size (染色体大小)

Background

For all living organisms, DNA replication is a key and highly regulated process of paramount importance for biological inheritance. The first step of DNA replication is the establishment of the genomic loci, where DNA synthesis begins. These loci are called replication origins (or just origins) (Méchali, 2010). In general, the beginning of DNA synthesis occurs after an initiator binds to an origin and recruits specific proteins that will result in the formation of the replisome in a process called origin firing. Each origin fired generates two replication forks that move in opposite directions. Replication forks synthesize DNA at a velocity (rate) that varies according to the organism and cell lineage (Myllykallio et al., 2000; Stanojcic et al., 2016; da Silva et al., 2017). The time needed for all chromosomes to replicate determines the S-phase duration, which seems to be robust for certain cell types and organisms (Zhang et al., 2017; da Silva et al., 2020).

Although prokaryotes from the Bacteria domain usually replicate their genome using only one origin, the vast majority of organisms (Archaea and Eukarya domains, in general) replicate their chromosomes from multiple origins (Leonard and Méchali, 2013). However, the exact number of origins fired per chromosome can vary according to cell type and the cellular environment (da Silva et al., 2020). In a recent study (da Silva et al., 2019), I developed a formula capable of estimating the minimum number of origins (MO) required to duplicate an entire chromosome within the S-phase duration. The principle of the formula is the bidirectional movement of the replication forks. Moreover, the S-phase duration, the size of the chromosome in question, and the average replication rate are required as parameters of the equation.

The estimation of MO per chromosome is an innovative method that allows the establishment of a threshold, which serves as a parameter to assist (or validate) genomic approaches that map origins, such as marker frequency analysis coupled to next-generation sequencing (MFA-seq), small leading nascent strand purification coupled to next-generation sequencing (SNS-seq), DNA microarray, and DNA combing. Under standard conditions, MO values show minimal variation since the variables used in the formula are, in general, stable. The MO formula is universal and can be applied for any organism, even procaryotes. In the analyzes presented here, the trypanosomatid parasite Trypanosoma brucei was used as a model. In this organism, the protocol typically takes 2-3 days from the estimation of the variables to results. However, if the necessary variables are available (i.e., they can be obtained from other studies), the estimation of MO per chromosome can be carried out immediately.

Materials and Reagents

  1. Microtubes 1.5 ml (Axygen, Maxyclear, catalog number: MCT-150-C-S )
  2. Centrifuge tubes 15 ml (Corning, catalog number: CLS430791 )
  3. Culture flasks 25 cm2 (Corning, canted neck, cap plug seal, catalog number: CLS430168 )
  4. Syringe filter 0.22 µm (Sartori, Minisart Syringe filter, catalog number: 16534 )
  5. Micropipette tips (Axygen, 10 µl, 200 µl and 1,000 µl)
  6. Serological pipettes (Costar Sterile, 10 ml)
  7. Microscope Slides (Knitell glass, non-color)
  8. Coverslips (Knitell glass, 22 x 22 mm)
  9. Combicoverslips (Genomic vision, catalog number: COV-002-RUO )
  10. Reusable plug modes (from Molecular combing system–Genomic vision)
  11. 6-well flat-bottom plate (Costar, catalog number: 38015 )
  12. Plastic coverslips (use a cut plastic pocket for binder)
  13. Nail varnish (any brand, preferably colorless)
  14. Click-iT EdU Cell Proliferation Kit, Alexa Fluor 488 dye (ThermoFisher Scientific, catalog number: C10337 )
  15. 5′-Iodo-2’-deoxyuridine (IdU) (Sigma-Aldrich, catalog number: I7125 )
  16. 5′-Chloro-2′-deoxyuridine (CldU) (Abcam, catalog number: ab213715 )
  17. Mouse α-BrdU/α-IdU monoclonal antibody (BD, catalog number: 347580 )
  18. Rat α-BrdU/α-CldU monoclonal antibody (Accurate, catalog number: YSRTMCA2060GA )
  19. Goat α-mouse IgG1 secondary antibody, Alexa Fluor 568 (ThermoFisher Scientific, catalog number: A-21124 )
  20. Goat α-rat IgG (H+L) secondary antibody, Alexa Fluor 488 (ThermoFisher Scientific, catalog number: A-11006 )
  21. Paraformaldehyde (Sigma-Aldrich, catalog number: 158127 )
  22. Poly-L-lysine hydrochloride (Sigma-Aldrich, catalog number: P2658 )
  23. Bovine serum albumin (Sigma-Aldrich, catalog number: 0 5470 )
  24. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  25. NaCl (Sigma-Aldrich, catalog number: S9888 )
  26. Na2HPO4 (Sigma Aldrich, catalog number: 255793 )
  27. Ethanol absolute (Merck Millipore, catalog number: 100983 )
  28. Vectashield Mounting Medium with DAPI (Vector Labs, catalog number: H-1200 )
  29. ProLong Gold Antifade Mountant (Thermo Fisher Scientific, catalog number: P36930 )
  30. Hydrochloric acid fuming 37% (Merck, catalog number: 100317 )
  31. Sodium hydroxide (Sigma-Aldrich, catalog number: 221465 )
  32. SDM-79 medium (LGC Biotecnologia, catalog number: BR30079-05 )
  33. Hemin (Merck, catalog number: H9039 )
  34. KCl (for molecular biology, any brand)
  35. KH2PO4 (for molecular biology, any brand)
  36. Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number: F7524 )
  37. Streptomycin sulfate salt (Sigma-Aldrich, catalog number: S6501 )
  38. Penicillin G sodium salt (Sigma-Aldrich, catalog number: P3032 )
  39. EDTA·2H2O (Ethylenediaminetetraacetic acid disodium salt dihydrate) (Sigma-Aldrich, catalog number: E5134 )
  40. NaOH (ACS reagent, any brand)
  41. Ethanol (Absolute, Merck, catalog number: 100983 )
  42. N-Laurylsarcosine sodium salt (Sigma-Aldrich, catalog number: L5125 )
  43. Proteinase K (ThermoFischer Scientific, catalog number: AM2544 )
  44. Anti-DNA Antibody, single stranded (mouse anti-ssDNA) (Millipore, catalog number: MAB3868 )
  45. Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody, Alexa Fluor 350 (anti-mouse Alexa Fluor 350) (ThermoFischer Scientific, catalog number: A-21140 )
  46. MES, free acid (ULTROL grade, Calbiochem, catalog number: 475893 )
  47. Phosphate buffered saline (1x PBS) (see Recipes)
  48. Hemin Solution (HS) (see Recipes)
  49. SDM-79 medium (for cultivate T. brucei procyclic cells) (see Recipes)
  50. 5′-chloro-2′-deoxyuridine solution (CldU-S) (see Recipes)
  51. 5-ethynyl-2’-deoxyuridine solution (EdU-S) (see Recipes)
  52. 5′-iodo-2′-deoxyuridine solution (IdU-S) (see Recipes)
  53. 0.5 M EDTA (see Recipes)
  54. 0.65 M EDTA (see Recipes)
  55. 1.5 M Tris-HCl (see Recipes)
  56. 1 M NaCl (see Recipes)
  57. 70% ethanol (see Recipes)
  58. 90% ethanol (see Recipes)
  59. DNA combing washing buffer (DC-WB) (see Recipes)
  60. DNA combing lysis buffer (DC-LB) (see Recipes)
  61. DNA combing blocking solution (DC-BS) (see Recipes)
  62. DNA combing denaturation buffer (DC-DB) (see Recipes)
  63. DNA combing primary antibodies solution (DC-PAS) (see Recipes)
  64. DNA combing secondary antibodies solution (DC-SAS) (see Recipes)
  65. DNA combing anti-ssDNA solution (DC-anti-ssDNA) (see Recipes)
  66. DNA combing Alexafluor 350 (DC-alexa350) (see Recipes)
  67. T10E1 buffer (see Recipes)
  68. 0.5 M MES buffer (see Recipes)
  69. Fixation buffer (FB) (see Recipes)
  70. Poly-L-lysine solution (PLS) (see Recipes)
  71. Permeabilization solution (PS) (see Recipes)

Equipment

  1. Microcentrifuge (Eppendorf, model: 5424 R )
  2. Motorized pipet dispenser (Fisher Scientific, Fisherbrand, catalog number: 03-692-172 )
  3. Water bath (Cientec, model: CT-226 )
  4. Magnetic stirrer (Fisatom, model: 753A )
  5. Incubator BOD (Vitrex, model: NI1705 )
  6. Fluorescence Microscope [Olympus, model: BX51 , coupled to an XM10 digital camera. Filters specifications: U-MWU2 (excitation = 330-385 nm; emission = 420 nm), U-MWIBA3 (excitation = 460-495 nm; emission = 510-550 nm), and U-MWG2 (excitation = 510-550 nm; emission = 590 nm)]
  7. Micropipettes (Gilson, models: Pipetman P10, P20, P200, and P1000)
  8. Centrifuge (Eppendorf, model: 5810 R ), equipped with 4 x 250 ml Swing-Bucket Rotor
  9. Neubauer chamber with cover glass (Sigma-Aldrich, model: Bright-LineTM Hemacytometer )
  10. Biosafety Class II A2 cabinet (Pachane, model: PA 700 )
  11. pH meter (Gehaka, model: PG1800 )
  12. Autoclave (Tomy Seiko, model: SS-245 )
  13. FiberComb–Molecular combing system (Genomic Vision, model: MCS-001 )

Software

  1. CeCyD (Butantan Institute, published in da Silva et al., 2020, http://cecyd.vital.butantan.gov.br/)
  2. Doubling Time Calculator (2006) (version 3.1.0, https://doubling-time.com/compute_more.php)
  3. Olympus Cell F software (Olympus, version 5.1.2640)
  4. ImageJ (NIH, version 1.47t)
  5. Microsoft Excel (Microsoft Office-any version) or GraphPad Prism (GraphPad software, Inc.)

Procedure

Notes:

  1. If you already have the values for chromosomes size, S-phase duration, and replication rate for the organism in question, the following steps (A-C) are not necessary.
  2. The description of the steps B (estimation of the S-phase) and C (estimation of the replication rate) uses as example the organism T. brucei. However, for other cell types these parameters can be found in specific studies when available [e.g., S. cerevisiae (Brewer et al., 1984; Sekedat et al., 2010; Ivanova et al., 2020); mammalian cells–MEFs cell (Ishida et al., 2001; Stanojcic et al., 2016; Pereira et al., 2017)].

  1. Obtaining values for chromosome size
    First option (for any organism):
    1. Access the website https://www.ncbi.nlm.nih.gov/.
    2. In ‘popular resources’ option (right side), click on genome.
    3. Type the name of the organism of interest.
    4. Obtain the values of chromosome size from the table.
    Second option (only for trypanosomatids organisms):
    1. Access the website https://tritrypdb.org/tritrypdb/https://tritrypdb.org/tritrypdb/.
    2. In ‘search for other data types’ option (middle column), click on ‘genomic sequences’, then 'organism’.
    3. Choose the organism of interest (e.g., Trypanosoma brucei).
    4. Click on ‘Get Answer’.
    5. Obtain the values of chromosome size (length) from the table.

  2. Estimation of the S-phase duration using CeCyD software
    CeCyD is a user-friendly website able to calculate the values of cytokinesis (C), mitosis (M), G2, S, and G1 phases of the cell cycle, for any organism. For this, the user must enter the following parameters on the website: doubling time, percentage of cells in cytokinesis, percentage of cells in mitosis, minimum time to detect two EdU-labeled nuclei in the same cell, percentage of cells EdU-labeled after EdU pulse, and the duration of this EdU pulse. The following steps of this section describe how these parameters are obtained in T. brucei (used as an example). However, for other cell types, the S-phase duration can be obtained directly from specific studies when available [e.g., S. cerevisiae (Brewer et al., 1984; Ivanova et al., 2020); mammalian cells – MEFs cells (Ishida et al., 2001; Pereira et al., 2017)].

    1. Prepare a set of T. brucei culture (here, it was used the procyclic forms) to estimate de doubling time. Initiates a growth curve with 1 x 106 cells/ml at 28 °C in 10 ml of culture.
      Note: The cell density for the beginning of the curve varies according to the cell type.
    2. Harvest cell samples and count daily, until it reached the stationary phase.
    3. Insert the values counted on https://doubling-time.com and take note of the doubling time value.
    4. Prepare a new set of T. brucei culture (procyclic forms) to estimate the percentage of cells in mitosis and cytokinesis. Incubate cells (28 °C) until they reach exponential phase (~1 x 107 cells/ml).
      Note: The cell density for the exponential phase varies according to the cell type used.
    5. Harvest 1 ml of the culture by centrifugation at 800 x g for 5 min at 4 °C and wash twice using 1 ml of 1x PBS.
    6. Remove the 1x PBS from cells carefully to preserve the pellet.
    7. Suspend the pellet in 1 ml of Fixation Buffer (FB) and transfer to a 1.5 ml microcentrifuge tube.
    8. Incubate at 4 °C for 7 min and wash three times using 1 ml of cold 1x PBS (centrifuging at 800 x g for 5 min in each wash). For the last wash, suspend the pellet carefully into 500 μl of 1x PBS. If the pellet is too small (i.e., almost invisible to the naked eye), decrease the volume to 100 μl of 1x PBS.
    9. Prepare the slides to receive the cells by spreading 2.5 μl of Poly-L-lysine solution (PLS) onto slide surface until PLS dry out. Prepare three slides for each sample from Step B8.
      Note: Use a coverslip to spread PLS onto slide (see da Silva et al., 2018 for more detail).
    10. Spread the suspended-pellet (from Step B8) carefully in each of the three slides. Use 25-30 μl and save the remaining suspended-pellet volume in case you need to remake some slides.
      Notes:
      1. Use the same surface where PLS was previous spread.
      2. Each slide is one technical replicate.
    11. Wait for the cells to precipitate and settle on slide for 10-15 min at room temperature. Ensure that the cells do not dry out.
    12. Permeabilize the cells by adding 50 μl of permeabilization solution (PS) for 10 min at room temperature.
    13. Wash the slide containing cells three times using 1x PBS.
      Note: Use a P1000 micropipette to spread (by sneezing) 1x PBS (1 ml) onto slide three times.
    14. Ensure all the liquid was removed from the slide surface containing cells and add 4 μl of Vectashield mounting medium containing DAPI.
      Note: This reagent is used as anti-fade mounting solution and to stain organelles containing DNA.
    15. Add a glass coverslip and seal using colorless nail varnish. Wait the varnish dry out for 5 min. The slide can be analyzed under a fluorescence microscope immediately or stored at 4 °C up to one month.
    16. Count at least 100 cells and identify cells in mitosis (with the nucleus in division) and cytokinesis (with two separated nuclei in the same cell). Figure 1 shows representative images of T. brucei cells in mitosis (A) and cytokinesis (B).
      Note: Capture images using the differential interference contrast (DIC) (if available) or phase contrast. Merge with DAPI to assist in the identification of cells in mitosis/cytokinesis.


      Figure 1. Representative images showing T. brucei cells in mitosis (left) and cytokinesis (right). It is worth to mentioning that in mitosis, the nucleus is dividing, while in cytokinesis the nucleus has already been divided. DIC was used to show the morphology of the cells, while DAPI was used to stain organelles containing DNA [nucleus (N) and kinetoplast (K)]. Scale bars = 10 μm.

    17. Take note the percentage of cells in mitosis and citokynesis.
    18. Prepare a new set of T. brucei culture to estimate the minimum time to detect two EdU-labeled nuclei in the same cell. Incubate cells (28 °C) until they reach exponential phase (~1 x 107 cells/ml).
      Note: The cell density for the exponential phase varies according to the cell type used.
    19. Add 100 µM of 5-ethynyl-2’-deoxyuridine solution (EdU-S) in the culture containing T. brucei cells and wait for 30 min.
    20. Harvest 500 µl of the culture by centrifugation (800 x g for 3 min at 4 °C) and wash twice using 1 ml of 1x PBS.
    21. Follow the Steps B6-B7 exactly as previously described.
    22. Incubate at 4 °C.
    23. Continue collecting samples every 15 min until reach 3 h of EdU incorporation (11 samples). Follow the Steps B20-B22 for each sample.
      Notes:
      1. Each sample should be incubated in FB for, at least, 7 min on ice.
      2. At the end of this step, there should be 12 samples collected every 15 min.
    24. Take the 12 samples and wash three times each using 1 ml of cold 1x PBS (centrifuging at 800 x g for 5 min in each wash). For the last wash, suspend the pellet of each sample carefully into 500 μl of 1x PBS. If the pellet is too small (i.e., almost invisible to the naked eye), decrease the volume to 100 μl of 1x PBS.
    25. Prepare 12 slides by spreading 2.5 μl of PLS onto slide surface until it dry out.
      Note: Use a coverslip to spread PLS onto slide. For more details, see da Silva et al. (2018).
    26. Spread the suspended-pellets (from Step B23) carefully in each slide. Use 25-30 μl and save the remaining volume in case you need to remake some slides.
      Note: Use the same surface where PLS was previous spread.
    27. Wait for the cells to decant and settle on slide for 10-15 min at room temperature. Ensure that the cells do not dry out.
    28. Permeabilize each sample by adding 50 μl of PS for 10 min at room temperature.
    29. Wash each slide containing cells three times using 1x PBS.
      Note: Use a P1000 micropipette to spread (by sneezing) 1x PBS (1 ml) onto slide three times.
    30. Follow the protocol of Click-iT EdU Imaging Kit, established by the manufacturer.
      Note: The protocol can be accessed here, on the topic Documents/Manuals and protocols:
      https://www.thermofisher.com/order/catalog/product/C10337#/C10337.
    31. At the end of the protocol established by the manufacturer, ensure all the liquid was removed from the slide surface containing cells and add 4 μl of Vectashield mounting medium containing DAPI.
      Note: This solution is used as the anti-fade mounting solution and to stain organelles containing DNA.
    32. Add a glass coverslip and seal using colorless nail varnish. Wait the varnish dry out for 5 min. The slide can be analyzed under a fluorescence microscope immediately or stored at 4 °C up to one month.
    33. Search (in each sample) for a parasite containing two EdU-labeled nuclei in the same cell (i.e., cell in citokynesis). Start from the shortest time sample, i.e., 15 min after 30 min of EdU incorporation, and proceed with the other samples (i.e., 30 min after 30 min of EdU incorporation, 45 min after 30 min of EdU incorporation, etc.).
      Note: For more details, see the description and images on Figure 1D from da Silva et al. (2019).
    34. Once observed, take note the time required for this. This is the minimum time to detect two EdU-labeled nuclei in the same cell.
      Note: The time obtained must be the sum of the initial time for EdU incorporation (30 min) plus the shortest time in which the two EdU-labeled nuclei were observed. In our case, this time was 2 h. For details, see da Silva et al. (2019).
    35. Prepare a new set of T. brucei culture to estimate the percentage of EdU-labeled cells during a EdU pulse. Incubate cells (28 °C) until they reach exponential phase (~1 x 107 cells/ml).
      Note: The cell density for the exponential phase varies according to the cell type used.
    36. Add 100 µM of EdU-S in the culture containing T. brucei cells and wait for 1 h.
      Note: This will be the duration of the EdU pulse, i.e., 1 h.
    37. Harvest 500 µl of the culture by centrifugation (800 x g for 3 min at 4 °C) and wash twice using 1 ml of 1x PBS.
    38. Follow the Steps B6-B13 exactly as previously described.
    39. Follow the protocol of Click-iT EdU Imaging Kit, established by the manufacturer.
      Note: The protocol can be accessed here, on the topic Documents/Manuals and protocols:
      https://www.thermofisher.com/order/catalog/product/C10337#/C10337.
    40. At the end of the protocol established by the manufacturer, ensure all the liquid was removed from the slide surface containing cells and add 4 μl of Vectashield mounting medium containing DAPI.
    41. Add a glass coverslip and seal using colorless nail varnish. Wait the varnish dry out for 5 min. The slide can be analyzed under a fluorescence microscope immediately or stored at 4 °C up to one month.
    42. Count at least 100 cells (total) and identify cells EdU-labeled. Figure 2 shows representative images of T. brucei EdU-positive cells.
    43. Measure the percentage of cells EdU-positive.
    44. Once you have all the required parameters, access the CeCyD website: http://cecyd.vital.butantan.gov.br/, and input the values to obtain the duration of all the cell cycle phases. Take note of the S-phase duration.


      Figure 2. Representative image showing EdU-positive T. brucei cells. The representative image is organized in four squares: DIC (indicating the morphology of the cells), DAPI (staining organelles containing DNA), EdU (showing cells that uptaked EdU during the S-phase), and Merged (overlay between DAPI and EdU). The white arrows indicate EdU-positive cells. Scale bar = 10 μm.

  3. Estimation of the replication rate using DNA combing
    The following steps of this section describe how the replication rate is obtained in T. brucei (used as an example). However, for other cell types, the replication rate can be obtained directly from specific studies when available [e.g., S. cerevisiae (Sekedat et al., 2010); mammalian cells–MEFs cells (Stanojcic et al., 2016)].

    1. Prepare a set of T. brucei culture (here, it was used the procyclic forms) to perform the DNA combing assay. Initiates the culture with 2 x 106 cells/ml in 10 ml of culture. Incubate at 28 °C for 24 h. Count the cell density. The culture must be in the concentration of ~1-2 x 107 cells/ml, totalizing ~1-2 x 108 cells.
      Note: The cell density for the beginning of the DNA combing assay varies according to the cell type.
    2. Add 300 µM of 5′-iodo-2′-deoxyuridine solution (IdU-S) and wait for 30 min.
    3. Add 500 µM of 5′-chloro-2′-deoxyuridine solution (CldU-S) and wait for 30 min.
      Note: Do not wash between the halogenated thymidine analogs (IdU and CldU) incorporation.
    4. Harvest the whole culture by centrifugation (1,600 x g for 3 min at 4 °C) and wash twice using 1 ml of 1x PBS. In the second wash, transfer the suspended-pellet to a 1.5 ml microcentrifuge tube.
    5. Wash using 500 µl of DNA combing washing buffer (DC-WB) and suspend the pellet in 50 µl of DC-WB. Keep at room temperature.
    6. Prepare 2% agarose low-melting: Weigh 10 mg of low melting agarose in a 1.5 ml centrifuge tube. Using another 1.5 ml centrifuge tube, heat 500 µl of DC-WB (90 °C for 5 min). Resuspend the 10 mg of low melting agarose using the heated 500 µl of DC-WB. Mix well to solubilize the agarose. Keep the solution heated at 65 °C.
    7. Prepare the reusable plug modes to receive 100 µl of the agarose + cells mixture.
    8. Gently mix 50 µl of the agarose solution (from Step C6) in the 50 µl of resuspended pellet (from Step C5), totalizing 100 µl. Add immediately to the previously prepared reusable plug model (from Step C7). Wait to cool and incubate at 4 °C for 1 h.
    9. Carefully unmold the plug in a 1.5 ml epp containing 500 µl of DNA combing lysis buffer (DC-LB).
    10. Incubate for 24 h at 50 °C.
    11. Replace the DC-LB (gently) by a new one (500 µl) and incubate for another 24 h at 50 °C.
    12. Wash twice using 1 ml of T10E1 buffer for 1 h (each wash) at room temperature.
    13. Add 1 ml of T10E1 buffer (gently) and incubate overnight at 4 °C.
    14. Discard the T10E1 buffer and incubate the plug in 800 µl of 0.5 M MES, pH 5.5 at 68 °C for 20 min. Next, remove the tube containing the plug from 68 °C and incubate it immediately at 42 °C for 10 min.
    15. Add 2 μl of β-agarase I (1,000 units/ml) and incubate overnight at 42 °C.
      Note: In this step, add the β-agarase I in 200 µl of 0.5 M MES. Mix well and add it carefully in the plug dissolved with 800 µl 0.5 M MES.
    16. Prepare the molecular combing system by adding 1 ml of 0.5 M MES into the reservoir of the FiberComb apparatus. Next, carefully pour the tube containing the plug dissolved (from Step C15) into the reservoir containing 1 ml of 0.5 M MES.
      Note: At this stage, it is normal to form a viscosity in the liquid coming from the tube.
    17. Immediately, place the Combicoverslip on the FiberComb apparatus and stretch the DNA according to equipment's instructions.
      Note: The solution containing the plug dissolved (i.e., incorporated DNA) inside the reservoir can be stored up to 1 week at 4 °C protected from light.
    18. After stretch the DNA onto Combicoverslip, let it dry for a few seconds and incubate it at 65 °C for 2-4 h.
    19. Place the Combicoverslip containing the DNA stretched in an appropriate container (e.g., use a 6-well flat-bottom plate). Carefully add 3 ml of DNA combing denaturation buffer (DC-DB) and incubate for 20 min.
    20. Wash three times using 1 ml of 1x PBS. Dehydrate the coverslip containing the DNA stretched successively using 2 ml of: 70% ethanol, then 90% ethanol, and 100% ethanol (1 min each). Remove the alcohol and let the Combicoverslip dry completely at room temperature.
    21. Block using 3 ml of DNA combing blocking solution (DC-BS) for 20 min at 37 °C.
    22. Prepare the DNA combing primary antibodies solution (DC-PAS).
      Note: The DC-PAS must be freshly prepared, as it is for immediate use.
    23. Add 20 µl of DC-PAS on the top of the Combicoverslip containing the DNA stretched (from Step C21). Cover with plastic coverslip. Incubate at 37 °C for 1 h protected from light.
    24. Wash twice using 1x PBS for 3 min each.
    25. Prepare the DNA combing secondary antibodies solution (DC-SAS).
      Note: The DC-SAS must be freshly prepared, as it is for immediate use.
    26. Add 20 µl of DC-SAS on the top of the Combicoverslip containing the DNA stretched (from Step C24). Cover with a plastic coverslip. Incubate at 37 °C for 1 h protected from light.
    27. Wash twice using 1x PBS for 3 min each.
    28. Prepare the DNA combing anti-ssDNA solution (DC-anti-ssDNA).
      Note: The DC-anti-ssDNA must be freshly prepared, as it is for immediate use.
    29. Add 20 µl of DC-anti-ssDNA on the top of the coverslip containing the DNA stretched (from Step C27). Cover with plastic coverslip. Incubate at 37 °C for 2 h protected from light.
    30. Wash twice using 1x PBS for 3 min each.
    31. Prepare the DNA combing Alexafluor 350 (DC-alexa350).
      Note: The DC-alexa350 must be freshly prepared, as it is for immediate use.
    32. Add 20 µl of DC-alexa350 on the top of the coverslip containing the DNA stretched (from Step C30). Cover with plastic coverslip. Incubate at 37 °C for 1 h protected from light.
    33. Wash twice using 1x PBS for 3 min each.
    34. Ensure all the liquid was removed from the coverslip containing the DNA stretched. Mount the coverslip on a slide adding 20 µl of ProLong Gold antifade. Seal using colorless nail varnish and let dry for at least 1 h at room temperature.
    35. Acquire images of DNA fibers using a fluorescence microscope. Merge acquired images using the Cell F Olympus or ImageJ software.
      Notes: 
      1. The observation of longer DNA fibers requires the capture of adjacent fields.
      2. The stretching factor along each molecule is contant (1 µm = 2 kb).
      3. Fibers < 100 kb must be excluded from the analysis to improve the accuracy of the assay.
      4. Only green tracks (containing CldU, the second analog added) continuing from red tracks (containing IdU, the first analog added) or blue tracks (non-incorporated DNA) must be used to determine replication rate.
      5. The length of the green track must be measured from intact green fibers: blue-green-red (BGR), red-green-blue (RGB), and blue-green-red-green-blue (BGRGB) events.
    36. Estimate the average replication rate using the formula:



      where v is the average replication rate (in kb.min-1), n is the number of DNA fibers analyzed, and t is the time of CldU pulse (in min).
      Note: At least three independent assays must be performed to improve the accuracy of the assay

Data analysis

  1. Data analysis
    With the possession of all variables (parameters) obtained previously, i.e., chromosome size, S-phase duration, and average replication rate, insert them in the formula and calculate the MO per chromosome analyzed.


    of note, N is the size of the chromosome in question, v is the average replication rate, and S is the S-phase duration.
    Note: If the right-hand side of the formula results in a fraction of a unit, then the next higher integer unit must be taken as the result of the formula, which is represented by the ceiling function (⌈ ⌉).

  2. Prepare a table to add the MO values found and compare them with the number of replication origins estimated/mapped by other approaches (e.g., DNA combing, MFA-seq analysis). In the current protocol, Table 1 was prepared for T. brucei and Table 2 for S. cerevisiae, which were used to demonstrate that the MO estimation can be applied in any organism.

    Table 1. Comparison among the MO, origins mapped by MFA-seq technique (Tiengwe et al., 2012), and origins estimated by DNA combing (Stanojcic et al., 2016) in T. brucei

    S-phase duration = 138.6 min (da Silva et al., 2019), replication rate = 1.84 kb·min-1 (Stanojcic et al., 2016). The values presented in this table were originally published in (da Silva et al., 2019).

    Table 2. Comparison among the MO, known replication origins (Nieduszynski et al., 2006), and origins estimated by DNA combing (Lengronne et al., 2001) in S. cerevisiae

    S-phase duration = 30 min (Brewer et al., 1984; Ivanova et al., 2020), replication rate = 1.6 kb·min-1 (Sekedat et al., 2010). The values presented in this table were originally published in (da Silva et al., 2020).

  3. Set up a graph in order to show trend lines for each approach analyzed (Figure 3 was made for T. brucei and Figure 4 for S. cerevisiae).


    Figure 3. Comparative analysis among different approaches used to estimate origins in T. brucei. The graph shows positive correlations between chromosome size and the number of origins estimated by: MO (black), origins estimated by MFA-seq (blue), and origins estimated by DNA combing (red). The trend lines for the approaches, as well as the equations, are shown. Studies are referenced in the legend of the graph. The data presented in this graph were originally published in da Silva et al. (2019).


    Figure 4. Comparative analysis among different approaches used to estimate origins in S. cerevisiae. The graph shows positive correlations between chromosome size and the number of origins estimated by: MO (black), known replication origins (green), and origins estimated by DNA combing (red). The trend lines for the approaches, as well as the equations, are shown. Studies are referenced in the legend of the graph. The data presented in this graph were originally published in da Silva et al. (2020).

        It is possible to observe an expected positive correlation between the number of origins and the size of each chromosome, i.e., the larger the chromosome, the more origins are required to replicate it within the S-phase duration. The MO allows the establishment of a threshold that can serve as a parameter by other methods. For instance, in T. brucei (Figure 3), when comparing the MO values with the MFA-seq ones, it is clear that some chromosomes are not able to be duplicated only with the origins mapped by MFA-seq, i.e., some blue circles are above from the threshold established by the MO values (black dotted line). Briefly, the reason is due MFA-seq approach has low resolution and map mainly constitutive (frequent) origins (da Silva et al., 2019 and 2020). Further analysis and a complete explanation can be found in the original publication from which this method was derived (da Silva et al., 2019).

Notes

  1. General notes
    1. If the values for the chromosomes size, S-phase duration, and replication rate are already available in the literature (i.e., they can be acquired from other studies), the Steps A-C described in ‘procedure’ are not necessary.
    2. The number of DNA fibers (n) analyzed in step c described on procedure must be as high as possible, since the higher the n, the more accurate the estimation of the replication rate will be.

  2. Technical tips
    1. To avoid false positives during the estimation of EdU-positive cells (Step B from procedure), do not overexpose fluorescence images during acquisition step.
    2. The incubation time of the halogenated thymidine analogs (IdU and CldU) varies according to the doubling-time (Step C from procedure) and it can be optimized for other cell types. The same is valid for the incubation time of the EdU (EdU pulse) (Step B from procedure).
    3. If it is possible, perform the whole protocol of DNA combing (Step C from procedure) in the dark, because DNA with halogenated thymidine analogs incorporated is more sensitive to the light.

Recipes

  1. Phosphate buffered saline (1x PBS)
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    2 mM KH2PO4
    1. Prepare the buffer by adding 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 in 800 ml of destilled water
    2. Adjust the pH to 7.4 with HCl
    3. Complete the volume to 1 L with destilled water
    4. Dispense the solution into aliquots (e.g., 250 ml) and sterilize by autoclaving (20 min, 120 °C, liquid cycle)
    5. Store at room temperature for up to six months
    6. Check the pH after prolonged use
  2. Hemin Solution (HS)
    50 mg/ml Hemin
    1. Prepare the solution by adding 50 mg of Hemin and 1 ml of 1 M NaOH in 80 ml of MilliQ water
    2. Stir vigorously on a magnetic stirrer
    3. Bring up the volume to 100 ml with MilliQ water
    4. Sterilize by filtering (using 0.22 μm filter)
    5. Store at 4 °C for up to one month
  3. SDM-79 medium (for cultivate T. brucei procyclic cells)
    90% (v/v) SDM-79 medium (incomplete)
    10% (v/v) heat-inactivated Fetal Bovine Serum (FBS)
    7.5 µg/ml Hemin
    0.134 µg/ml Streptomycin sulfate salt
    0.060 µg/ml Penicillin G sodium salt
    1. Prepare the medium by adding 50 ml of heat-inactivated FBS, 75 µl of HS, 67 mg of Streptomycin sulfate salt, and 30 mg of Penicillin G sodium salt in 400 ml of SDM-79 medium (incomplete)
    2. Adjust the pH to 7.3 and complete the volume to 500 ml with SDM-79 medium (incomplete)
    3. Sterilize by filtering (using 0.22 μm filter) and dispense into aliquots (e.g., 100 ml)
    4. Store at 4 °C for up to one month
  4. 5′-chloro-2′-deoxyuridine solution (CldU-S)
    10 mM of CldU diluted in water
    1. Prepare the buffer by adding 26.26 mg of 5′-chloro-2′-deoxyuridine in 9 ml of water
    2. Solubilizes and complete the volume to 10 ml
    3. Sterilize by filtering (using 0.22 μm syringe filter) and dispense the solution into aliquots (e.g., 1 ml)
    4. Store at -20 °C for up to six months
  5. 5-ethynyl-2’-deoxyuridine solution (EdU-S)
    10 mM of EdU diluted in 1x PBS
    1. Prepare the solution by adding 2 ml of 1x PBS to component A from Click-iT® EdU Imaging Kit
    2. Mix well to solubilize it
    3. Sterilize by filtering (using 0.22 μm syringe filter) and dispense the solution into aliquots (e.g., 1 ml)
    4. Store at -20 °C for up to six months
  6. 5′-iodo-2′-deoxyuridine solution (IdU-S)
    10 mM of IdU diluted in water alkalinized
    Note: IdU is not readily soluble at physiological pH.
    1. Prepare the buffer by adding 35.4 mg of 5′-iodo-2′-deoxyuridine in 5 ml of alkalinized MilliQ water (adjust the Milli-Q water pH to 9.5 using 5 M NaOH with the aid of a pH meter)
    2. Solubilize and complete the volume to 10 ml with Milli-Q water
    3. Sterilize by filtering (using 0.22 μm syringe filter) and dispense the solution into aliquots (e.g., 1 ml)
    4. Store at -20 °C for up to six months
  7. 0.5 M EDTA, pH 8.0
    1. Prepare the solution by adding 186.1 g of disodium EDTA·2H2O to 800 ml of distilled H2
    2. Stir vigorously on a magnetic stirrer while adjust the pH to 8.0 with NaOH (~20 g of NaOH pellets)
    3. Bring up the volume to 1 L with distilled water
    4. Dispense into aliquots (e.g., 250 ml) and sterilize by autoclaving
    5. Store at room temperature for up to six months
  8. 0.65 M EDTA, pH 8.0
    1. Prepare the solution by adding 24.2 g of disodium EDTA·2H2O to 80 ml of distilled H2
    2. Stir vigorously on a magnetic stirrer while adjust the pH to 8.0 with NaOH
    3. Bring up the volume to 100 ml with distilled water
    4. Sterilize by autoclaving
    5. Store at room temperature for up to one month
  9. 1.5 M Tris-HCl, pH 8.8
    1. Prepare the solution by adding 181.65 g Tris base in 800 ml of MilliQ water
    2. Adjust the pH to 8.8 using concentrated HCl
    3. Bring up the volume to 1 L with MilliQ water
    4. Dispense into aliquots (e.g., 250 ml) and sterilize by autoclaving
    5. Store at room temperature for up to six months
  10. 1 M NaCl
    1. Prepare the solution by adding 58.44 g of NaCl in 800 ml of distilled H2
    2. Stir vigorously on a magnetic stirrer (heat to help dissolve)
    3. Bring up the volume to 1 L with distilled water
    4. Dispense into aliquots (e.g., 250 ml) and sterilize by autoclaving
    5. Store at room temperature for up to one year
  11. 70% ethanol
    1. Prepare the solution by adding 35 ml of ethanol absolute in 15 ml of distilled H2O
    2. Mix well and store at 4 °C for up to one year
  12. 90% ethanol
    1. Prepare the solution by adding 45 ml of ethanol absolute in 5 ml of distilled H2O
    2. Mix well and store at 4 °C for up to one year
  13. DNA combing washing buffer (DC-WB)
    100 mM EDTA, pH 8.0
    10 mM Tris-HCl
    20 mM NaCl 
    1. Prepare the buffer by adding 20 ml of 500 mM EDTA, 666 µl of Tris-HCl, and 2 ml of NaCl in 77.33 ml of MilliQ water
    2. Mix well and sterilize by filtering (using 0.22 μm filter)
    3. Store at 4 °C for up to one month
  14. DNA combing lysis buffer (DC-LB)
    0.65 EDTA, pH 8.0
    1% N-Laurylsarcosine
    100 μg/ml Proteinase K
    1. Prepare the buffer by adding 50 µg of N-Laurylsarcosine and 500 µg of proteinase K in 4 ml of 0.65 M EDTA, pH 8.0
    2. Mix well and bring up the volume to 5 ml using 0.65 M EDTA, pH 8.0
    3. Store at room temperature up to one week
  15. DNA combing blocking solution (DC-BS)
    1x PBS
    1% Bovine Serum Albumin (BSA)
    0.1% Triton X-100
    1. Prepare the solution by adding 100 µg of BSA and 10 µl of Triton X-100 in 9,880 µl of 1x PBS
    2. Mix well and sterilize by filtering (using 0.22 μm filter)
    3. Store at 4 °C up to one week
  16. DNA combing denaturation buffer (DC-DB)
    0.5 M NaOH
    1 M NaCl
    1. Prepare the solution by adding 200 mg of NaOH and 58.4 mg of NaCl in 8 ml of distilled H2O
    2. Mix well and bring up the volume to 10 ml using distilled H2O
    3. Store at room temperature up to one week
  17. DNA combing primary antibodies solution (DC-PAS)
    1/10 anti-IdU (mouse anti-BrdU)
    1/10 anti-CldU (rat anti-BrdU)
    100% DC-BS
    1. Prepare the solution by adding 2 µl of anti-IdU, 2 µl of anti-CldU in 16 µl of DC-BS
    2. Mix well and use immediately
  18. DNA combing secondary antibodies solution (DC-SAS)
    1/20 anti-mouse Alexafluor 568
    1/20 anti-rat Alexafluor 488
    100% DC-BS
    1. Prepare the solution by adding 1 µl of anti-mouse Alexafluor 568, 1 µl of anti-rat Alexafluor 488 in 18 µl of DC-BS
    2. Mix well and use immediately
  19. DNA combing anti-ssDNA solution (DC-anti-ssDNA)
    1/5 anti-ssDNA (mouse anti-ssDNA)
    100% DC-BS
    1. Prepare the solution by adding 4 µl of anti-ssDNA in 16 µl of DC-BS
    2. Mix well and use immediately
  20. DNA combing Alexafluor 350 (DC-alexa350)
    1/10 anti-mouse Alexafluor 350
    100% DC-BS
    1. Prepare the solution by adding 2 µl of anti-mouse Alexafluor 350 in 18 µl of DC-BS
    2. Mix well and use immediately
  21. T10E1 buffer
    10 mM Tris-HCl, pH 8.8
    1 mM EDTA, pH 8.0
    1. Prepare the buffer by mixing 66.6 µl of 1.5 M Tris-HCl, 20 µl of 0.5 M EDTA, and 9,913 µl of MilliQ H2O
    2. Mix well and sterilize by filtering (using 0.22 μm filter)
    3. Store at 4 °C up to one month
  22. 0.5 M MES buffer, pH 5.5
    1. Prepare the solution by adding 9.76 g of MES free acid in 80 ml of distilled H2O
    2. Stir vigorously on a magnetic stirrer
    3. Adjust the pH to 5.5 using NaOH
    4. Bring up the volume to 100 ml using distilled H2O
    5. Sterilize by autoclaving
    6. Store at room temperature for up to one month
  23. Fixation buffer (FB)
    4% (w/v) Paraformaldehyde in 1x PBS
    1. Prepare the buffer by adding 2.1 g of Paraformaldehyde (powder, 95%) in 40 ml 1x PBS
    2. Solubilize and complete the volume to 50 ml with 1x PBS
    3. Store at 4 °C for up to one month
  24. Poly-L-lysine solution (PLS)
    0.1% (w/v) Poly-L-Lysine hydrochloride diluted in water
    1. Prepare the buffer by adding 10 mg of Poly-L-Lysine hydrochloride in 9 ml of water
    2. Solubilize and complete the volume to 10 ml with water
    3. Sterilize by filtering (using 0.22 μm syringe filter) and dispense the solution into aliquots (e.g., 1 ml)
    4. Store at 4 °C for up to six months
  25. Permeabilization solution (PS)
    1. Prepare the buffer by adding 10 μl of Triton X-100 in 9,990 μl of 1x PBS
    2. Mix well, sterilize by filtering (using 0.22 μm syringe filter), and dispense the solution into aliquots (e.g., 1 ml)
    3. Store at 4 °C for up to six months

Acknowledgments

This work has been derived from da Silva et al. (2019) and da Silva et al. (2020). The development of the protocol presented here was supported by São Paulo Research Foundation (FAPESP) – Center of Toxins, Immune Response and Cell Signaling (CeTICS) under grants 2014/24170-5 and 2017/18719-2. Currently, Marcelo S. da Silva is fellow from FAPESP (grants 2019/10753-2 and 2020/10277-3).

Competing interests

The author declares that there is no conflict of interest regarding the publication of this article.

References

  1. Brewer, B.J., Chlebowicz-Sledziewska, E., Fangman, W.L. (1984). Cell cycle phases in the unequal mother/daughter cell cycles of Saccharomyces cerevisiae. Mol Cell Biol 4: 2529-2531.
  2. da Silva, M. S., Marin, P. A., Repolês, B. M., Elias, M. C. and Machado, C. R. (2018). Analysis of DNA Exchange Using Thymidine Analogs (ADExTA) in Trypanosoma cruzi. Bio-protocol 8(24): e3125.
  3. da Silva, M. S., Cayres-Silva, G. R., Vitarelli, M. O., Marin, P. A., Hiraiwa, P. M., Araujo, C. B., Scholl, B. B., Avila, A. R., McCulloch, R., Reis, M. S. and Elias, M. C. (2019). Transcription activity contributes to the firing of non-constitutive origins in African trypanosomes helping to maintain robustness in S-phase duration. Sci Rep 9(1): 18512.
  4. da Silva, M. S., Pavani, R. S., Damasceno, J. D., Marques, C. A., McCulloch, R., Tosi, L. R. O. and Elias, M. C. (2017). Nuclear DNA replication in trypanosomatids: There are no easy methods for solving difficult problems. Trends Parasitol 33(11): 858-874.
  5. da Silva, M. S., Vitarelli, M. O., Souza, B. F. and Elias, M. C. (2020). Comparative analysis of the minimum number of replication origins in trypanosomatids and yeasts. Genes (Basel) 11(5).
  6. Ishida, S., Huang, E., Zuzan, H., Spang, R., Leone, G., West, M., and Nevins, J. R. (2001). Role for E2F in control of both DNA replication and mitotic functions as revealed from DNA microarray analysis. Mol Cell Biol 21(14): 4684-4699.
  7. Ivanova, T., Maier, M., Missarova, A., Ziegler-Birling, C., Carey, L.B., Mendoza, M. (2020). Budding yeast complete DNA replication after chromosome segregation begins. Nat Commun 11(1): 2267.
  8. Lengronne, A., Pasero, P., Bensimon, A., Schwob E. (2001). Monitoring S phase progression globally and locally using BrdU incorporation in TK+ yeast strains. Nucleic Acids Res 2001, 29(7): 1433-1442.
  9. Leonard, A. C. and Mechali, M. (2013). DNA replication origins. Cold Spring Harb Perspect Biol 5(10): a010116.
  10. Mechali, M. (2010). Eukaryotic DNA replication origins: many choices for appropriate answers. Nat Rev Mol Cell Biol 11(10): 728-738.
  11. Myllykallio, H., Lopez, P., Lopez-Garcia, P., Heilig, R., Saurin, W., Zivanovic, Y., Philippe, H. and Forterre, P. (2000). Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon. Science 288(5474): 2212-2215.
  12. Nieduszynski, C.A., Knox, Y., Donaldson, A.D. (2006). Genome-wide identification of replication origins in yeast by comparative genomics. Genes Dev 20(14): 1874-1879.
  13. Pereira, P. D., Serra-Caetano, A., Cabrita, M., Bekman, E., Braga, J., Rino, J., Santus, R., Filipe, P. L., Sousa, A. E., and Ferreira, J. A. (2017). Quantification of cell cycle kinetics by EdU (5-ethynyl-2’-deoxyuridine)-coupled-fluorescence-intensity analysis. Oncotarget 8(25): 40514-40532.
  14. Sekedat, M. D., Fenyö, D., Rogers, R. S., Tackett, A. J., Aitchison, J. D. and Chait, B. T. (2010). GINS motion reveals replication fork progression is remarkably uniform throughout the yeast genome. Mol Syst Biol 6: 353.
  15. Stanojcic, S., Sollelis, L., Kuk, N., Crobu, L., Balard, Y., Schwob, E., Bastien, P., Pages, M. and Sterkers, Y. (2016). Single-molecule analysis of DNA replication reveals novel features in the divergent eukaryotes Leishmania and Trypanosoma brucei versus mammalian cells. Sci Rep 6: 23142.
  16. Tiengwe, C., Marcello, L., Farr, H., Dickens, N., Kelly, S., Swiderski, M., Vaughan, D., Gull, K., Barry, J. D., Bell, S. D. and McCulloch, R. (2012). Genome-wide analysis reveals extensive functional interaction between DNA replication initiation and transcription in the genome of Trypanosoma brucei. Cell Rep 2(1): 185-197.
  17. Zhang, Q., Bassetti, F., Gherardi, M. and Lagomarsino, M. C. (2017). Cell-to-cell variability and robustness in S-phase duration from genome replication kinetics. Nucleic Acids Res 45(14): 8190-8198.

简介

[摘要] 比率可能因多种因素而变化,但最主要的因素是复制应力。一些研究应用了不同的方法来估计复制源在不同生物体中的数量和位置。然而,如果没有一个参数来限制必要起源的最小值,那么不太敏感的技术可能会产生相互矛盾的结果。估计每个染色体的最小复制源数量(MO)是一种创新的方法,它允许建立一个阈值,作为绘制起源的基因组方法的参数。为此,MO可以很容易地通过一个公式得到,这个公式需要作为参数:染色体大小、S期持续时间和复制率。在基因组数据库(如NCBI)中可以获得任何生物体的染色体大小,通过监测DNA复制来估计S期的持续时间,并通过DNA组合方法获得复制率。 提供了一种简单、快速的估算MO的方法一个新的方法学框架来协助研究任何有机体。
关键词: DNA复制,复制来源,复制率,S期持续时间,染色体大小

[背景] 对于所有生物来说,DNA复制是一个关键的、高度调控的过程,对生物遗传至关重要。DNA复制的第一步是建立基因组基因座,DNA合成由此开始。这些基因座被称为复制起源(或仅仅是起源)(Méchali,2010)。一般来说,DNA合成的开始发生在引发剂与起始点结合并招募特定蛋白质后,这将导致在称为原点激发的过程中形成复制体。每个激发的源都生成两个向相反方向移动的复制分叉。复制叉合成DNA的速度(速率)随生物体和细胞谱系的不同而变化(Myllykallio,2000;Stanojcic,2016;da Silva,2017)。所有染色体复制所需的时间决定了S期的持续时间,这似乎对某些细胞类型和生物体来说是很稳健的(Zhang,2017年;da Silva,2020年)。等等。等等。等等。等等。等等。
尽管绝大多数真核生物和原核生物(2013年)的染色体都是从真核生物和真核生物中复制出来的(尽管大多数真核生物和原核生物仅在2013年复制)。然而,每个染色体的确切起源数目可能因细胞类型和细胞环境而异(da Silva,2020年)。在最近的一项研究(da Silva,2019)中,我开发了一个公式,能够估算出在S期持续时间内复制整个染色体所需的最小起源数(MO)。该公式的原理是复制叉的双向移动。此外,S期持续时间、染色体大小和平均复制率都是方程的参数。等等。等等。
每个染色体的MO估计是一种创新的方法,它允许建立一个阈值,作为一个参数来帮助(或验证)绘制起源的基因组方法,例如标记频率分析与下一代测序(MFA-seq)相结合,小型领先的新生链纯化与下一代测序(SNS-seq)、DNA微阵列和DNA梳理相结合。在标准条件下,MO值显示最小的变化,因为公式中使用的变量通常是稳定的。MO公式具有普遍性,适用于任何生物体,甚至原核生物。在这里介绍的分析中,锥虫被用作模型。在这种情况下,从变量估计到结果,方案通常需要2-3天的时间。然而,如果有必要的变量(,它们可以从其他研究中获得),每个染色体的钼的估计可以立即进行。布氏锥虫即

关键字:DNA复制, 复制起源点, 复制速度, S期持续时间, 染色体大小

材料和试剂
 
1.      1.5 ml微型管(Axygen,Maxyclear,目录号:MCT-150-C-S)
2.      离心管(目录号:S791)
3.      25 cm2培养瓶(康宁,斜颈,盖塞密封,目录号:CLS430168)
4.      注射器过滤器0.22µm(Sartori,Miniart注射器过滤器,目录号:16534)
5.      微量移液管尖端(Axygen、10µl、200µl和1000µl)
6.      血清学移液管(Costar无菌,10 ml)
7.      显微镜载玻片(刀形玻璃,非彩色)
8.      盖玻片(针状玻璃,22 x 22毫米)
9.      Combicoverslips(基因组视觉,目录号:COV-002-RUO)
10.   可重复使用的插头模式(来自分子梳理系统-基因组视觉)
11.   6井平底板(Costar,目录号:38015)
12.   塑料盖玻片(使用切割的塑料袋作为粘合剂)
13.   指甲油(任何牌子,最好是无色)
14.   Click iT EdU细胞增殖试剂盒,Alexa Fluor 488染料(ThermoFisher Scientific,目录号:C10337)
15.   5'-碘-2'-脱氧尿苷(IdU)(Sigma-Aldrich,目录号:I7125)
16.   5′-氯-2′-脱氧尿苷(CldU)(Abcam,目录号:ab213715)
17.   小鼠α-BrdU/α-IdU单克隆抗体(BD,目录号:347580)
18.   大鼠α-BrdU/α-CldU单克隆抗体(准确,目录号:YSRTMCA2060GA)
19.   第二代小鼠IgA-218号,Alexa-218-科学抗体
20.   山羊α-大鼠IgG(H+L)二级抗体,Alexa Fluor 488(ThermoFisher Scientific,目录号:A-11006)
21.   多聚甲醛(Sigma-Aldrich,目录号:158127)
22.   聚赖氨酸盐酸盐(Sigma-Aldrich,目录号:P2658)
23.   牛血清白蛋白(Sigma-Aldrich,目录号:05470)
24.   Triton-T87 Sigma目录
25.   氯化钠(Sigma-Aldrich,目录号:S9888)
26.  编号:HP572O93
27.  无水乙醇(默克密理博,目录号:100983)
28.  带DAPI的Vectashield安装介质(Vector Labs,目录号:H-1200)
29.  延长金防腐蚀贴片(赛默飞世尔科学公司,目录号:P36930)
30.  盐酸烟化37%(默克公司,目录号:100317)
31.  氢氧化钠(Sigma-Aldrich,目录号:221465)
32.  SDM-79培养基(LGC生物技术,目录号:BR30079-05)
33.  海明(默克,产品目录号:H9039)
34.  KCl(用于分子生物学,任何品牌)
35.   KH2PO4(用于分子生物学,任何品牌)
36.   胎牛血清(FBS)(Sigma-Aldrich,目录号:F7524)
37.   硫酸链霉素盐(Sigma-Aldrich,目录号:S6501)
38.   青霉素G钠盐(Sigma-Aldrich,目录号:P3032)
39.   EDTA·2H2O(乙二胺四乙酸二钠二水合物)(Sigma-Aldrich,目录号:E5134)
40.   NaOH(ACS试剂,任何品牌)
41.   乙醇(无水,默克,目录号:100983)
42.   N-月桂酰肌氨酸钠盐(Sigma-Aldrich,目录号:L5125)
43.   蛋白酶K(ThermoFischer Scientific,目录号:AM2544)
44.   抗DNA抗体,单链(小鼠抗ssDNA)(Millipore,目录号:MAB3868)
45.   山羊抗鼠IgG2b交叉吸附二级抗体,Alexa Fluor 350(抗鼠Alexa Fluor 350)(Thermofisher Scientific,目录号:A-21140)
46.   MES,游离酸(ULTROL级,Calbiochem,目录号:475893)
47.  磷酸盐缓冲盐水(1x PBS)(见配方)
48.  氯化血红素溶液(HS)(见配方)
49.  SDM-79培养基(用于培养布氏原环细胞)(见配方)
50.  5′-氯-2′-脱氧尿苷溶液(CldU-S)(见配方)
51.  5-乙炔基-2'-脱氧尿苷溶液(EdU-S)(见配方)
52.  5′-碘-2′-脱氧尿苷溶液(IdU-S)(见配方)
53.  50万EDTA(见配方)
54.  0.65 M EDTA(见配方)
55.  1.5 M Tris HCl(见配方)
56.  1 M NaCl(见配方)
57.  70%乙醇(见配方)
58.  90%乙醇(见配方)
59.  DNA梳理洗涤缓冲液(DC-WB)(见配方)
60.  DNA结合裂解缓冲液(DC-LB)(见配方)
61.  DNA梳理阻断液(DC-BS)(见配方)
62.  DNA结合变性缓冲液(DC-DB)(见配方)
63.  DNA结合初级抗体溶液(DC-PAS)(见配方)
64.  DNA结合二级抗体溶液(DC-SAS)(见配方)
65.  DNA结合抗ssDNA溶液(DC抗ssDNA)(见配方)
66.  DNA梳理Alexafluor350(DC-alexa350)(见配方)
67.  T10E1缓冲液(见配方)
68.  0.5 M MES缓冲器(见配方)
69.  固定缓冲液(FB)(见配方)
70.  聚赖氨酸溶液(PLS)(见配方)
71.  渗透溶液(PS)(见配方)
 
使用的设备
 
1.      微型离心机(Eppendorf,型号:5424R)
2.      电动移液管分配器(Fisher Scientific,Fisherbrand,目录号:03-692-172)
3.      水浴(Cientec,型号:CT-226)
4.      磁力搅拌器(Fisatom,型号:753A)
5.      培养箱BOD(玻璃体,型号:NI1705)
6.      荧光显微镜【Olympus,型号:BX51,与XM10数码相机相连。滤光片规格:U-MWU2(激发=330-385 nm;发射=420 nm);U-MWIBA3(激发=460-495 nm;发射=510-550 nm);U-MWG2(激发=510-550 nm;发射=590 nm)]
7.      微量移液管(Gilson,型号:移液管P10、P20、P200和P1000)
8.      离心机(Eppendorf,型号:5810 R),配有4 x 250 ml摆斗转子
9.      带盖玻璃的Neubauer室(Sigma-Aldrich,型号:Bright LineTM血细胞仪)
10.   生物安全二级A2柜(Pachane,型号:PA 700)
11.   pH计(Gehaka,型号:PG1800)
12.   高压灭菌器(Tomy Seiko,型号:SS-245)
13.   FiberComb–分子梳理系统(Genomic Vision,型号:MCS-001)
 
软件
 
1.      CeCyD(丁烷研究所,发表于da Silva等人,2020年,http://cecyd.vital.butatan.gov.br/)
2.      倍增时间计算器(2006)(3.1.0版,https://doubling-time.com/compute_more.php)
3.      Olympus Cell F软件(Olympus,版本5.1.2640)
4.      NIH 47J版
5.      Microsoft Excel(Microsoft Office任意版本)或GraphPad Prism(GraphPad软件公司)
 
程序
 
笔记:
1.     如果你已经有了有关生物体的染色体大小、S期持续时间和复制率的值,那么下面的步骤(A-C)就没有必要了。
2.     步骤B(S期的估计)和C(复制率的估计)的描述以布鲁氏杆菌为例。然而,对于其他细胞类型,这些参数可在特定研究中找到(如酿酒酵母)(Brewer等人,1984年;Sekedat等人,2010年;Ivanova等人,2020年);哺乳动物细胞——MEFs细胞(Ishida等人,2001年;Stanojcic等人,2016年;Pereira等人,2017年)]。
 
A、 获得染色体大小值
第一种选择(对于任何生物体):
1.      访问网站https://www.ncbi.nlm.nih.gov/。
2.     在“流行资源”选项(右侧),点击基因组。
3.     键入感兴趣的有机体的名称。
4.     从表中获得染色体大小值。
 
第二种选择(仅适用于锥虫类生物):
1.      访问网站https://tritrypdb.org/tritrypdb/。
2.      在“搜索其他数据类型”选项(中间一栏),单击“基因组序列”,然后单击“有机体”。
3.      选择感兴趣的生物体(如布氏锥虫)。
5.      单击“获取答案”。
6.      从表中获得染色体大小(长度)值。
 
B、 用CeCyD软件估计S相持续时间
CeCyD是一个用户友好的网站,可以计算细胞周期的胞质分裂(C)、有丝分裂(M)、G2、S和G1期的数值。为此,用户必须在网站上输入以下参数:倍增时间、胞质分裂细胞百分率、有丝分裂细胞百分率、同一细胞中检测两个EdU标记细胞核的最短时间、EdU脉冲后EdU标记细胞的百分比以及该EdU脉冲的持续时间。本节的以下步骤描述了如何在T.brucei中获得这些参数(用作示例)。然而,对于其他细胞类型,S期持续时间可直接从特定研究中获得(如酿酒酵母(Brewer等人,1984年;Ivanova等人,2020年);哺乳动物细胞——MEFs细胞(Ishida等人,2001年;Pereira等人,2017年)]。
 
1.     准备一套布氏木霉培养物(这里,它被用于前环形式)来估计去加倍时间。在10毫升培养液中,28°C条件下,以1 x 106个细胞/毫升开始生长曲线。
注:曲线开头的单元格密度随单元格类型而变化。
2.     采集细胞样本并每天计数,直到达到固定期。
3.     插入计数的值https://doubling-time.com和注意倍增时间值。
4.     准备一套新的布鲁氏杆菌培养物(前环型),以估计有丝分裂和胞质分裂中的细胞百分比。培养细胞(28°C),直到达到指数期(~1 x 107个细胞/ml)。
注:指数相的电池密度因所用电池类型而异。
5.     在4°C下,以800 x g离心5分钟,收获1 ml培养物,并使用1 ml 1x PBS洗涤两次。
6.     小心地从细胞中取出1x PBS以保存颗粒。
7.     将颗粒悬浮在1 ml固定缓冲液(FB)中,并转移至1.5 ml微型离心管中。
8.     在4°C下培养7分钟,并使用1毫升冷1x PBS清洗三次(每次清洗以800 x g离心5分钟)。最后一次清洗时,小心地将颗粒悬浮在500μl 1x PBS中。如果颗粒太小(即肉眼几乎看不见),则将体积减小至100μl 1x PBS。
9.     将2.5μl聚赖氨酸溶液(PLS)涂在载玻片表面,直到PLS干燥,制备载玻片以接收细胞。为步骤B8中的每个样品准备三张幻灯片。
注:使用盖玻片将PLS铺在载玻片上(更多详情见da Silva等人,2018年)。
10.  从每一步的玻片(B8)中小心地将小球(B8)分散。使用25-30μl并保存剩余悬浮颗粒体积,以备需要重新制作一些载玻片。
笔记:
a。使用与PLS之前相同的表面。
b。每张幻灯片都是一个技术复制品。
11.  在室温下等待细胞沉淀并在载玻片上沉淀10-15分钟。确保电池不会干燥。
12.  通过在室温下添加50μl渗透溶液(PS)10分钟使电池渗透。
13.  用1x PBS清洗含有细胞的载玻片三次。
注:用P1000微量吸管(通过打喷嚏)将1x PBS(1毫升)涂在载玻片上三次。
14.  确保从含有细胞的载玻片表面清除所有液体,并添加4μl含有DAPI的Vectashield安装介质。
注:本试剂用作防褪色贴装液,用于染色含有DNA的细胞器。
15.  添加玻璃盖玻片和密封使用无色指甲油。等待清漆干燥5分钟。可以立即在荧光显微镜下分析载玻片,也可以在4°C下保存一个月。
16.  计数至少100个细胞,识别有丝分裂(核分裂)和胞质分裂(同一细胞内有两个分离的细胞核)的细胞。图1显示有丝分裂(A)和胞质分裂(B)中布鲁氏杆菌细胞的代表性图像。
注:使用差分干涉对比度(DIC)(如果可用)或相位对比度拍摄图像。与DAPI合并以帮助识别有丝分裂/胞质分裂中的细胞。
 


图1。代表性图片显示布鲁氏杆菌细胞有丝分裂(左)和胞质分裂(右)。值得一提的是,有丝分裂时,细胞核正在分裂,而在胞质分裂中,细胞核已经分裂。DIC显示细胞形态,DAPI染色含DNA的细胞器[细胞核(N)和动细胞(K)]。比例尺=10μm。
 
17.  注意有丝分裂和城市化中细胞的百分比。
18.  准备一套新的布氏木霉培养物,估计在同一细胞中检测两个EdU标记的细胞核的最短时间。培养细胞(28°C),直到达到指数期(~1 x 107个细胞/ml)。
注:指数相的电池密度因所用电池类型而异。
19.  将100µM 5-乙炔基-2'-脱氧尿苷溶液(EdU-S)加入含有布氏锥虫细胞的培养物中,并等待30分钟。
20.  通过离心法收获500µl培养物(800 x g,4°C下3分钟),并使用1 ml 1x PBS洗涤两次。
21.  完全按照前面描述的步骤B6-B7进行操作。
22.  在4°C下培养。
23.  继续每15分钟采集一次样品,直到EdU加入3小时(11个样品)。对于每个样品,遵循步骤B20-B22。
笔记:
a。每个样品应在FB中冰上培养至少7分钟。
b。在该步骤结束时,每15分钟应采集12个样本。
24.  取12个样品,用1ml冷1x PBS清洗三次(每次清洗以800 x g离心5分钟)。最后一次清洗时,小心地将每个样品的颗粒悬浮在500μl 1x PBS中。如果颗粒太小(即肉眼几乎看不见),则将体积减小至100μl 1x PBS。
25.  在载玻片表面涂抹2.5μl PLS,直至其干燥,制备12个载玻片。
注意:请使用盖玻片将其铺在载玻片上。更多详细信息,请参见dasilva等人。(2018年)。
26.  将悬浮颗粒(从步骤B23开始)小心地摊铺在每个载玻片中。使用25-30μl并保存剩余体积,以备需要重新制作一些幻灯片。
注:请使用与PLS之前相同的表面。
27.  在室温下等待电池倒流并在载玻片上静置10-15分钟。确保电池不会干燥。
28.  在室温下每50μl的温度下加入渗透性PS。
29.  用1x PBS清洗每个含有细胞的载玻片三次。
注:用P1000微量吸管(通过打喷嚏)将1x PBS(1毫升)涂在载玻片上三次。
30.  遵循制造商制定的Click-iT EdU成像套件协议。
注意:协议可以在这里访问,主题是文件/手册和协议:
https://www.thermofisher.com/order/catalog/product/C10337#/C10337.
31.  在制造商制定的方案结束时,确保从含有电池的载玻片表面清除所有液体,并添加4μl含有DAPI的Vectashield安装介质。
注:此溶液用作防褪色贴装液,用于染色含有DNA的细胞器。
32.  添加玻璃盖玻片和密封使用无色指甲油。等待清漆干燥5分钟。可以立即在荧光显微镜下分析载玻片,也可以在4°C下保存一个月。
33.  (在每个样本中)搜索同一细胞(即citokynesis细胞)中含有两个EdU标记细胞核的寄生虫。从最短时间的样本开始,即加入EdU 30分钟后15分钟,然后继续进行其他样本(即加入EdU 30分钟后30分钟,加入EdU 30分钟后45分钟,等等)。
注:有关更多详细信息,请参见da Silva等人在图1D中的描述和图像。(2019年)。
34.  一旦观察到,请记下所需的时间。这是检测同一细胞中两个EdU标记的细胞核的最短时间。
注:获得的时间必须是EdU掺入的初始时间(30min)加上观察到两个EdU标记核的最短时间之和。在我们的例子中,这个时间是2小时。有关详细信息,请参阅dasilva等人。(2019年)。
35.  准备一套新的布鲁氏杆菌培养物,以估计EdU脉冲期间EdU标记细胞的百分比。培养细胞(28°C),直到达到指数期(~1 x 107个细胞/ml)。
注:指数相的电池密度因所用电池类型而异。
36.  在含有布氏锥虫细胞的培养液中加入100µM的EdU-S并等待1小时。
注:这将是EdU脉冲的持续时间,即1小时。
37.  通过离心法收获500µl培养物(800 x g,4°C下3分钟),并使用1 ml 1x PBS洗涤两次。
38.  完全按照前面描述的步骤B6-B13进行操作。
39.  遵循制造商制定的Click-iT EdU成像套件协议。
注意:协议可以在这里访问,主题是文件/手册和协议:
https://www.thermofisher.com/order/catalog/product/C10337#/C10337.
40.  在制造商制定的方案结束时,确保从含有电池的载玻片表面清除所有液体,并添加4μl含有DAPI的Vectashield安装介质。
41.  添加玻璃盖玻片和密封使用无色指甲油。等待清漆干燥5分钟。可以立即在荧光显微镜下分析载玻片,也可以在4°C下保存一个月。
42.  计数至少100个细胞(总共),并确定细胞EdU标记。图2显示了布鲁氏杆菌EdU阳性细胞的代表性图像。
43.  检测EdU阳性细胞百分率。
44.  一旦您获得了所有必需的参数,请访问CeCyD网站:http://cecyd.vital.butatan.gov.br/,并输入值以获得所有细胞周期阶段的持续时间。注意S相持续时间。
 


图2。显示EdU阳性brucei细胞的代表性图像。染色显示细胞间有组织的DNA(DAPI-DAPI),显示细胞间有组织的DNA(DAPI-DAPI)。白色箭头表示EdU阳性细胞。比例尺=10μm。
 
C、 用DNA梳合法估计复制率
本节的以下步骤描述了如何在T.brucei中获得复制率(用作示例)。然而,对于其他细胞类型,可直接从特定研究中获得复制率(如酿酒酵母(Sekedat等人,2010年);哺乳动物细胞——MEFs细胞(Stanojcic等人,2016年)】。
 
1.     准备一套布氏锥虫培养物(这里,它是使用前环形式)来进行DNA组合分析。在10 ml培养液中用2 x 106细胞/ml开始培养。28℃孵育24小时,计数细胞密度。培养物的浓度必须为~1-2×107个细胞/ml,总共约为1-2×108个细胞。
注:DNA结合试验开始时的细胞密度因细胞类型而异。
2.     加入300µM的5′-碘-2′-脱氧尿苷溶液(IdU-S)并等待30分钟。
3.     添加500µM 5′-氯-2′-脱氧尿苷溶液(CldU-S),并等待30分钟。
注:不要在卤化胸苷类似物(IdU和CldU)合并之间洗涤。
4.     通过离心法收获整个培养物(1600 x g,4°C下3分钟),并使用1 ml 1x PBS洗涤两次。在第二次清洗中,将悬浮颗粒转移到1.5 ml微型离心管中。
5.     使用500µl DNA结合洗涤缓冲液(DC-WB)洗涤,并将颗粒悬浮在50µl DC-WB中。保持室温。
6.     制备2%低熔点琼脂糖:称取10mg低熔点琼脂糖于1.5ml离心管中。使用另一根1.5 ml离心管,加热500µl DC-WB(90°C,5分钟)。使用加热后的500µl DC-WB重新悬浮10 mg低熔点琼脂糖。混合均匀,使琼脂糖溶解。将溶液加热至65°C。
7.     准备可重复使用的塞子模式,以接收100µl琼脂糖+细胞混合物。
8.     将50µl琼脂糖溶液(来自步骤C6)轻轻混合在50µl再悬浮颗粒(步骤C5)中,总计100µl。立即添加到先前准备好的可重复使用的塞子模型中(从步骤C7开始)。待冷却,在4°C下孵育1h。
9.     在含有1.5μl的DNA的缓冲液中仔细地进行分离。
10.  在50°C下培养24小时。
11.  将DC-LB(轻轻地)替换为新的DC-LB(500µl),并在50°C下再培养24小时。
12.  在室温下用1ml T10E1缓冲液清洗两次(每次清洗)1h。
13.  加入1ml T10E1缓冲液(轻轻地)并在4°C下培养过夜。
14.  丢弃T10E1缓冲液,并在800µl 0.5 M MES(68°C下pH 5.5)中培养20分钟。然后,从68°C取出含有塞子的试管,并立即在42°C下培养10分钟。
15.  加入2μlβ-琼脂酶I(1000单位/ml),并在42°C下培养过夜。
注意:在此步骤中,添加β-200µl 0.5 M MES中的琼脂酶I。混合均匀,小心地将其添加到用800µl 0.5 M MES溶解的塞子中。
16.  将1ml 0.5mmes加入纤维束装置的储液罐中,制备分子梳理系统。接下来,小心地将含有溶解塞子的试管(从步骤C15)倒入含有1 ml 0.5 M MES的储液罐中。
注意:在这个阶段,从管子出来的液体中形成粘度是正常的。
17.  立即,将Combicoverslip放在FiberComb仪器上,并根据设备的指示拉伸DNA。
注:含有塞子的溶液在储液罐内溶解(即,并入DNA)可在4°C避光保存1周。
18.  将DNA拉伸到Combicoverslip上后,让其干燥几秒钟,并在65°C下培养2-4小时。
19.  将含有DNA拉伸后的混合物放在适当的容器中(例如,使用6孔平底板)。小心加入3ml DNA结合变性缓冲液(DC-DB),孵育20分钟。
20.  用1毫升1x PBS清洗三次。用2ml:70%乙醇、90%乙醇和100%乙醇(各1分钟)使含有DNA的盖玻片脱水。除去酒精,让Combicoverslip在室温下完全干燥。
21.  在37°C下用3 ml DNA结合阻断液(DC-BS)阻断20分钟。
22.  制备DNA结合一级抗体溶液(DC-PAS)。
注意:DC-PAS必须是新制备的,因为它可以立即使用。
23.  将20µl DC-PAS添加到含有延伸DNA的Combicoverslip顶部(从步骤C21开始)。用塑料盖玻片盖住。在37°C下避光培养1h。
24.  用1x PBS清洗两次,每次3分钟。
25.  制备DNA结合二级抗体溶液(DC-SAS)。
注意:DC-SAS必须是新制备的,因为它可以立即使用。
26.  将20µl DC-SAS添加到含有拉伸DNA的Combicoverslip顶部(从步骤C24开始)。用塑料盖玻片盖住。在37°C下避光培养1h。
27.  用1x PBS清洗两次,每次3分钟。
28.  制备DNA结合抗ssDNA溶液(DC-anti-ssDNA)。
注意:DC抗ssDNA必须是新制备的,因为它可以立即使用。
29.  在含有拉伸DNA的盖玻片顶部添加20µl DC抗ssDNA(从步骤C27开始)。用塑料盖玻片盖住。在37°C下避光培养2小时。
30.  用1x PBS清洗两次,每次3分钟。
31.  准备DNA梳理Alexafluor350(DC-alexa350)。
注意:DC-alexa350必须是新制备的,因为它可以立即使用。
32.  将20µl DC-alexa350添加到包含拉伸DNA的盖玻片顶部(从步骤C30开始)。用塑料盖玻片盖住。在37°C下避光培养1h。
33.  用1x PBS清洗两次,每次3分钟。
34.  确保所有的液体都从含有DNA拉伸的盖玻片上取下。将盖玻片贴在玻片上,加入20µl长效金防腐蚀剂。用无色指甲油密封,室温下干燥至少1小时。
35.  用荧光显微镜获取DNA纤维的图像。使用Cell F Olympus或ImageJ软件合并采集的图像。
笔记:
a。观察较长的DNA纤维需要捕获相邻的区域。
b。沿着每个分子的拉伸因子是恒定的(1µm=2 kb)。
c.c。分析中必须排除<100 kb的纤维,以提高分析的准确性。
d。必须从包含第一个模拟轨道(IdU)中添加包含第一个模拟轨道(IdU)或包含第二个模拟轨道(IdU)的复制,以确定第二个包含蓝色轨道(IdU)的复制。
e。绿色轨迹的长度必须从完整的绿色光纤测量:蓝绿-红(BGR)、红-绿-蓝(RGB)和蓝-绿-红-绿-蓝(BGRGB)事件。
36.  使用以下公式估计平均复制速率:
 
(一)
 
其中v是平均复制速率(inkb.分钟-1) n为分析的DNA纤维数,t为CldU脉冲时间(分钟)。
注:必须至少进行三次独立的化验,以提高化验的准确性
 
数据分析
 
1.     并用前面得到的染色体复制率公式(i)计算各染色体的平均着丝粒数。
 
         (二)
 
问题是染色体复制的平均持续时间。N五S
注:如果公式右侧的结果是一个单位的分数,则下一个更高的整数单位必须作为公式的结果,它由上限函数表示(⌈ ⌉).
 
2.     准备一张表格来添加发现的MO值,并将其与其他方法(如DNA梳理、MFA序列分析)估计/映射的复制源数量进行比较。表1和表2中的AES可用于证明表1中的任何生物体。
 
表1。比较T.brucei中的MO、MFA-seq技术绘制的起源(Tiengwe等人,2012年)和DNA梳理法估计的起源(Stanojcic等人,2016年)

S期持续时间=138.6分钟(da Silva等人,2019年),复制率=1.84 kb·min-1(Stanojcic等人,2016年)。本表中的数值最初发表于(da Silva等人,2019年)。
 
表2。比较酿酒酵母中的MO、已知复制起源(Nieduszynski等人,2006年)和DNA梳理估算的来源(Lengronne等人,2001年)

S相持续时间=30分钟(Brewer等人,1984年;Ivanova等人,2020年),复制率=1.6 kb·min-1(Sekedat等人,2010年)。本表中的数值最初发表于(da Silva等人,2020年)。
 
3.     建立一个图表,以显示所分析的每种方法的趋势线(图3为布鲁氏杆菌,图4为酿酒酵母)。
 
图3。布鲁氏菌起源估计方法的比较分析。图中显示了染色体大小与来源数量之间的正相关:MO(黑色)、MFA-seq(蓝色)和DNA梳理(红色)估算的起源数量。给出了这些方法的趋势线和方程。图表图例中引用了研究结果。图中所示的数据最初发表在da Silva et al。(2019年)。
 
图4。用于估计酿酒酵母起源的不同方法的比较分析。图中显示了染色体大小与来源数量之间的正相关:MO(黑色)、已知复制来源(绿色)和DNA梳理估算的来源(红色)。给出了这些方法的趋势线和方程。图表图例中引用了研究结果。图中所示的数据最初发表在da Silva et al。(2020年)。
 
可以观察到起源数目与每条染色体大小之间的预期正相关,即染色体越大,在S期内复制它所需的起源越多。MO允许建立一个阈值,该阈值可以作为其他方法的参数。例如,在T.brucei(图3)中,当比较MO值与MFA-seq值时,很明显有些染色体不能只与MFA-seq所映射的原点重复,即有些蓝圈超出了MO值设定的阈值(黑色虚线)。简单地说,原因是由于MFA-seq方法的分辨率较低,并且绘制的主要是结构性(频繁)成因(da-Silva等人,2019年和2020年)。进一步的分析和完整的解释可以在衍生该方法的原始出版物中找到(da Silva et al.,2019)。
 
笔记
 
A、 一般注意事项
1.     如果染色体大小、S期持续时间和复制率的值已在文献中提供(即,可以从其他研究中获得),则“程序”中描述的步骤A-C是不必要的。
2.     程序中描述的步骤c中分析的DNA纤维(n)的数量必须尽可能高,因为n越高,对复制率的估计就越准确。
 
B、 技术提示
1.     为了避免在估计EdU阳性细胞的过程中出现假阳性(步骤B),采集步骤中不要过度曝光荧光图像。
2.     卤化胸苷类似物(IdU和CldU)的孵育时间根据倍增时间(程序步骤C)而变化,并且可以针对其他细胞类型进行优化。这同样适用于EdU(EdU脉冲)的培养时间(步骤B)。
3.     如果可能的话,在黑暗中进行DNA梳理的整个过程(步骤C),因为加入卤化胸苷类似物的DNA对光更敏感。
 
食谱
 
1.     磷酸盐缓冲盐水(1x PBS)
137毫米NaCl 2.7毫米KCl 10毫米Na2HPO4 2毫米KH2PO4
a、 将8g NaCl、0.2g KCl、1.44g Na2HPO4和0.24g KH2PO4加入800 ml蒸馏水中制备缓冲液
b、 用盐酸将pH调至7.4
c、 用蒸馏水使体积达到1L
d、 将溶液分为小份(如250毫升)并通过高压灭菌(20分钟,120°C,液体循环)进行灭菌
e、 在室温下储存6个月
f、 长时间使用后检查pH值
2.     氯化血红素溶液(HS)
50毫克/毫升氯化血红素
a、 将50 mg氯化血红素和1 ml 1 M NaOH加入80 ml MilliQ水中制备溶液
b、 用磁力搅拌器使劲搅拌
c、 用MilliQ水将体积增加到100毫升
d、 过滤灭菌(使用0.22μm过滤器)
e、 在4°C下储存一个月
3.     SDM-79培养基(用于培养布氏原环细胞)90%(v/v)SDM-79培养基(不完全)
10%(v/v)热灭活胎牛血清(FBS)
7.5微克/毫升氯化血红素
0.134微克/毫升硫酸链霉素盐0.060微克/毫升青霉素g钠盐
a、 通过在400 ml SDM-79培养基(不完整)中添加50 ml热灭活FBS、75µl HS、67 mg硫酸链霉素盐和30 mg青霉素G钠盐来制备培养基
b、 将pH调至7.3,并用SDM-79培养基(不完全)将体积补充至500毫升
c、 过滤灭菌(使用0.22μm过滤器)并分配到小份(例如100 ml)
d、 在4°C下储存一个月
4.     5′-氯-2′-脱氧尿苷溶液(CldU-S)
10 mM CldU在水中稀释
a、 将26.26 mg 5′-氯-2′-脱氧尿苷加入9 ml水中制备缓冲液
b、 溶解并使体积达到10毫升
c、 过滤消毒(使用0.22μm注射器过滤器),并将溶液分为小份(如1 ml)
d、 在-20°C下储存6个月
5.     5-乙炔基-2'-脱氧尿苷溶液(EdU-S)
在1x PBS中稀释10 mM EdU
a、 通过将2ml 1x PBS添加到Click-iT®EdU成像套件的组分a中,制备溶液
b、 混合均匀使其溶解
c、 过滤消毒(使用0.22μm注射器过滤器),并将溶液分为小份(如1 ml)
d、 在-20°C下储存6个月
6.     5′-碘-2′-脱氧尿苷溶液(IdU-S)
在碱化水中稀释10 mM的IdU
注:IdU在生理pH值下不易溶解。
a、 将35.4 mg 5′-碘-2′-脱氧尿苷加入5 ml碱化miliq水中制备缓冲液(使用5 M NaOH和pH计将Milli-Q水的pH值调整为9.5)
b、 溶解并用Milli-Q水将其溶解至10mL
c、 过滤消毒(使用0.22μm注射器过滤器),并将溶液分为小份(如1 ml)
d、 在-20°C下储存6个月
7.     0.5 M EDTA,pH值8.0
a、 在800 ml蒸馏水中加入186.1 g EDTA·2H2O二钠,制备溶液
b、 在磁力搅拌器上剧烈搅拌,同时用NaOH(约20 g NaOH颗粒)将pH调至8.0
c、 用蒸馏水将体积增加到1升
d、 分为小份(如250毫升)并通过高压灭菌灭菌
e、 在室温下储存6个月
8.     0.65 M EDTA,pH值8.0
a、 将24.2 g EDTA·2H2O二钠加入80 ml蒸馏水中制备溶液
b、 在磁力搅拌器上剧烈搅拌,同时用NaOH将pH调至8.0
c、 用蒸馏水将体积增加到100毫升
d、 高压灭菌器
e、 在室温下储存一个月
9.     1.5 M Tris HCl,pH值8.8
a、 将181.65 g Tris碱加入800 ml MilliQ水中制备溶液
b、 使用浓盐酸将pH值调节至8.8
c、 用MilliQ水将体积提高到1L
d、 分为小份(如250毫升)并通过高压灭菌灭菌
e、 在室温下储存6个月
10.  1M氯化钠
a、 在800 ml蒸馏水中加入58.44 g NaCl制备溶液
b、 在磁力搅拌器上用力搅拌(加热帮助溶解)
c、 用蒸馏水将体积增加到1升
d、 分为小份(如250毫升)并通过高压灭菌灭菌
e、 在室温下储存一年
11.  70%乙醇
a、 在15 ml蒸馏水中加入35 ml无水乙醇制备溶液
b、 充分搅拌,并在4°C下储存一年
12.  90%乙醇
a、 通过在5 ml蒸馏水中添加45 ml无水乙醇来制备溶液
b、 充分搅拌,并在4°C下储存一年
13.  DNA梳理洗涤液(DC-WB)
100毫米EDTA,pH值8.0
10毫米Tris HCl
20毫米氯化钠
a、 通过在77.33 ml MilliQ水中添加20 ml 500 mM EDTA、666µl Tris HCl和2 ml NaCl来制备缓冲液
b、 混合均匀,过滤灭菌(使用0.22μm过滤器)
c、 在4°c下储存一个月
14.  DNA结合裂解缓冲液(DC-LB)
0.65 EDTA,pH值8.0
1%N-月桂酰肌氨酸
100μg/ml蛋白酶K
a、 将50µg N-月桂酰肌氨酸和500µg蛋白酶K加入4 ml 0.65 M EDTA(pH 8.0)中制备缓冲液
b、 混合均匀,使用0.65 M EDTA(pH 8.0)将体积增至5 ml
c、 室温下储存一周
15.  DNA梳理阻断液(DC-BS)
1个PBS
1%牛血清白蛋白(BSA)
0.1%Triton X-100
a、 在9880µl 1x PBS中加入100µg BSA和10µl Triton X-100,制备溶液
b、 混合均匀,过滤灭菌(使用0.22μm过滤器)
c、 在4°c下储存一周
16.  DNA结合变性缓冲液(DC-DB)
0.5 M氢氧化钠
1M氯化钠
a、 在8 ml蒸馏水中加入200 mg NaOH和58.4 mg NaCl制备溶液
b、 混合均匀,用蒸馏水将体积增加到10毫升
c、 室温下储存一周
17.  DNA结合初级抗体溶液(DC-PAS)
1/10抗IdU(小鼠抗BrdU)
1/10抗CldU(大鼠抗BrdU)
100%直流-断路器
a、 通过在16µl DC-BS中添加2µl抗IdU和2µl抗CldU来制备溶液
b、 搅拌均匀,立即使用
18.  DNA结合二级抗体溶液(DC-SAS)
1/20抗鼠Alexafluor 568
Alexa 488抗氟鼠
100%直流-断路器
a、 通过在18µl DC-BS中添加1µl抗鼠Alexafluor 568和1µl抗鼠Alexafluro 488来制备溶液
b、 搅拌均匀,立即使用
19.  DNA结合抗ssDNA溶液(DC-anti-ssDNA)
1/5抗ssDNA(小鼠抗ssDNA)
100%直流-断路器
a、 通过在16µl DC-BS中添加4µl抗ssDNA来制备溶液
b、 搅拌均匀,立即使用
20.  DNA梳理Alexafluor350(DC-alexa350)
1/10抗鼠Alexafluor 350
100%直流-断路器
a、 通过在18µl DC-BS中添加2µl抗鼠Alexafluor 350来制备溶液
b、 搅拌均匀,立即使用
21.  T10E1缓冲器
10毫米Tris HCl,pH值8.8
1毫米EDTA,pH值8.0
a、 通过混合66.6µl 1.5 M Tris HCl、20µl 0.5 M EDTA和9913µl MilliQ H2O制备缓冲液
b、 混合均匀,过滤灭菌(使用0.22μm过滤器)
c、 在4°c下储存一个月
22.  0.5 M MES缓冲液,pH值5.5
a、 在80 ml蒸馏水中加入9.76 g MES游离酸制备溶液
b、 用磁力搅拌器使劲搅拌
c、 使用NaOH将pH值调整到5.5
d、 使用蒸馏水将体积增加到100毫升
e、 高压灭菌
f、 在室温下储存一个月
23.  固定缓冲液(FB)4%(w/v)多聚甲醛(1x PBS)
a、 在40 ml 1x PBS中加入2.1 g多聚甲醛(粉末,95%)制备缓冲液
b、 用1x PBS溶解并使体积达到50 ml
c、 在4°c下储存一个月
24.  聚赖氨酸溶液(PLS)0.1%(w/v)聚赖氨酸盐酸盐在水中稀释
a、 将10毫克聚赖氨酸盐酸盐加入9毫升水中制备缓冲液
b、 溶解并用水使体积达到10毫升
c、 过滤消毒(使用0.22μm注射器过滤器),并将溶液分为小份(如1 ml)
d、 在4°C下储存6个月
25.  渗透溶液(PS)
在1x PBS中稀释0.1%(v/v)Triton X-100
a、 通过在9990μl 1x PBS中添加10μl Triton X-100制备缓冲液
b、 混匀,过滤灭菌(使用0.22μm注射器过滤器),并将溶液分为小份(如1ml)
c、 在4°c下储存6个月
 
致谢
 
这项工作来源于dasilva等人。(2019年)和da Silva等人。(2020年)。本文提出的方案的制定得到了圣保罗研究基金会(FAPESP)-毒素、免疫反应和细胞信号中心(CeTICS)的资助,资助资金为2014/24170-5和2017/18719-2。目前,Marcelo S.da Silva是FAPESP的研究员(拨款2019/10753-2和2020/10277-3)。

相互竞争的利益
 
作者声明这篇文章的发表不存在利益冲突。
 
工具书类
 
1.      Brewer,B.J.,Chlebowicz Sledziewska,E.,Fangman,W.L.(1984年)。酿酒酵母Mol细胞生物学4:2529-2531不等母子细胞周期的细胞周期。.
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引用:da Silva, M. S. (2020). Estimation of the Minimum Number of Replication Origins Per Chromosome in any Organism. Bio-protocol 10(20): e3798. DOI: 10.21769/BioProtoc.3798.
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