155 lines
17 KiB
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155 lines
17 KiB
XML
<document id="04B6695EB3BE4F22BD1FE80A28FA900F" ID-DOI="10.1093/zoolinnean/zlad196" ID-ISSN="0024-4082" ID-Zenodo-Dep="14423105" IM.bibliography_approvedBy="carolina" IM.illustrations_approvedBy="carolina" IM.materialsCitations_approvedBy="felipe" IM.metadata_approvedBy="carolina" IM.tables_requiresApprovalFor="GgImagineBatch" IM.taxonomicNames_approvedBy="carolina" IM.treatments_approvedBy="carolina" checkinTime="1734029342775" checkinUser="plazi" docAuthor="Fu, Jinyu, Gao, Yunyi, Gao, Feng, Sheng, Yalan, Al-Farraj, Saleh A., Chen, Zigui & Wang, Chundi" docDate="2024" docId="D06287D4FFB2FF9EFC1E05A3FE87FEE3" docLanguage="en" docName="zlad196.pdf" docOrigin="Zoological Journal of the Linnean Society 202 (3)" docSource="https://doi.org/10.1093/zoolinnean/zlad196" docStyle="DocumentStyle:4F230B9370E98E256D973D6DFB57F36C.9:ZoolJLinnSoc.2023-.journal_article" docStyleId="4F230B9370E98E256D973D6DFB57F36C" docStyleName="ZoolJLinnSoc.2023-.journal_article" docStyleVersion="9" docTitle="Paramecium tetraurelia" docType="treatment" docVersion="1" lastPageNumber="9" masterDocId="2C5BFFACFFBAFF97FFDF0149FC7DFFE5" masterDocTitle="An overview of genetic manipulation tools in the studies of ciliates * with emphasis on gene knockout * knockdown * and overexpression" masterLastPageNumber="13" masterPageNumber="1" pageNumber="9" updateTime="1734439180940" updateUser="carolina">
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<mods:title id="5FDA28F59D35FB391DC88FF36F954133">An overview of genetic manipulation tools in the studies of ciliates * with emphasis on gene knockout * knockdown * and overexpression</mods:title>
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<mods:namePart id="5FAB2520C69AAD69F1295F2B23A836CF">Fu, Jinyu</mods:namePart>
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<mods:affiliation id="248DF2206D9C5C5FDE4572C773112D5A">Institute of Evolution & Marine Biodiversity * Ocean University of China * Qingdao 266003 * China & Key Laboratory of Evolution & Marine Biodiversity (Ministry of Education) * Ocean University of China * Qingdao 266003 * China</mods:affiliation>
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<mods:nameIdentifier id="C58A09EA75DC1CB338EA4CAF97855772" type="email">wangcd@sdu.&zigui.chen@cuhk.edu.hk</mods:nameIdentifier>
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<mods:namePart id="A1D1274DBE2C030FBC7C018535EEEE13">Gao, Yunyi</mods:namePart>
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<mods:affiliation id="246FB737002E9C6FDE6DDB03899E760E">Institute of Evolution & Marine Biodiversity * Ocean University of China * Qingdao 266003 * China & Key Laboratory of Evolution & Marine Biodiversity (Ministry of Education) * Ocean University of China * Qingdao 266003 * China</mods:affiliation>
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<mods:nameIdentifier id="785A950E6CB9AE3B1960341E2E75C6FE" type="email">wangcd@sdu.&zigui.chen@cuhk.edu.hk</mods:nameIdentifier>
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<mods:namePart id="7FE318DC68A9BCC9A3D403E103961F71">Gao, Feng</mods:namePart>
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<mods:affiliation id="593A308A277976F7AF85DB4E96484C24">Institute of Evolution & Marine Biodiversity * Ocean University of China * Qingdao 266003 * China & Key Laboratory of Evolution & Marine Biodiversity (Ministry of Education) * Ocean University of China * Qingdao 266003 * China</mods:affiliation>
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<mods:nameIdentifier id="19AA6C8065F774D196D6B48F6CDDA766" type="email">wangcd@sdu.&zigui.chen@cuhk.edu.hk</mods:nameIdentifier>
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<mods:namePart id="B7A73B27711D10576DCDF6F7535EECC5">Sheng, Yalan</mods:namePart>
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<mods:affiliation id="29179F76A0972E47DEF0CF220226B091">Guangdong Key Laboratory of Subtropical Biodiversity and Biomonitoring * School of Life Sciences * South China Normal University * Guangzhou 510631 * China</mods:affiliation>
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<mods:namePart id="AF15A32EEDB036686DA88D571DC5544D">Al-Farraj, Saleh A.</mods:namePart>
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<mods:affiliation id="A651FEFB44DB584B1EF2AB0323EF626F">Zoology Department * College of Science * King Saud University * Riyadh 11451 * Saudi Arabia</mods:affiliation>
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<mods:namePart id="D4608B4CD1D18B1B28339470EC48561A">Chen, Zigui</mods:namePart>
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<mods:affiliation id="1704EB832DB8ED2EA0247FB36141DF15">Institute of Evolution & Marine Biodiversity * Ocean University of China * Qingdao 266003 * China & Key Laboratory of Evolution & Marine Biodiversity (Ministry of Education) * Ocean University of China * Qingdao 266003 * China</mods:affiliation>
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<mods:nameIdentifier id="34040EEDBC8EC178FDA7C1AD86D25468" type="email">wangcd@sdu.&zigui.chen@cuhk.edu.hk</mods:nameIdentifier>
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<mods:namePart id="1ECCF76DD9536D1AF72559030DC7415F">Wang, Chundi</mods:namePart>
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<mods:affiliation id="4B63B34774278F9B6EAAD8ED61783F74">Laboratory of Marine Protozoan Biodiversity & Evolution * Marine College * Shandong University * Weihai 264209 * China</mods:affiliation>
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<mods:title id="9242E79FECCF1DE01DD2691E66225DC1">Zoological Journal of the Linnean Society</mods:title>
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<mods:date id="B0B6E0D4C266807EA700775DCC97D5F5">2024</mods:date>
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<mods:number id="7FB0CF783B57FC2AEBDDF1949D1A9787">2024-02-08</mods:number>
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<mods:number id="F5F1707F3ADA75C0CA65E0D60751511C">202</mods:number>
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<mods:url id="94D402BDBD2F344A65F1FAD21033ABA6">https://doi.org/10.1093/zoolinnean/zlad196</mods:url>
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<mods:identifier id="83CB8A20AA142203D60AA13578BE4E4E" type="DOI">10.1093/zoolinnean/zlad196</mods:identifier>
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<mods:identifier id="FF1AC442463D4F9865B163449A90F000" type="ISSN">0024-4082</mods:identifier>
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<mods:identifier id="1791EE7BA02218A698BBB71806130F66" type="Zenodo-Dep">14423105</mods:identifier>
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<subSubSection id="10D16549FFB2FF9FFC1E05A3F947FAE0" box="[961,1338,1258,1285]" pageId="8" pageNumber="9" type="nomenclature">
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<paragraph id="587436C2FFB2FF9FFC1E05A3F947FAE0" blockId="8.[825,1475,1258,2010]" box="[961,1338,1258,1285]" pageId="8" pageNumber="9">
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<emphasis id="6ABFEAD0FFB2FF9FFC1E05A3F947FAE0" bold="true" box="[961,1338,1258,1285]" pageId="8" pageNumber="9">
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Gene overexpression in
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<taxonomicName id="9FCB4D41FFB2FF9FFB6105A2F947FAE0" authority="Sonneborn" box="[1214,1338,1258,1285]" class="Hypotrichea" family="Oxytrichidae" genus="Paramecium" kingdom="Chromista" order="Oxytrichida" pageId="8" pageNumber="9" phylum="Ciliophora" rank="species" species="tetraurelia">
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<emphasis id="6ABFEAD0FFB2FF9FFB6105A2F947FAE0" bold="true" box="[1214,1338,1258,1285]" italics="true" pageId="8" pageNumber="9">P. tetraurelia</emphasis>
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</taxonomicName>
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</emphasis>
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</paragraph>
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<subSubSection id="10D16549FFB2FF9EFCE60458FE87FEE3" lastPageId="9" lastPageNumber="10" pageId="8" pageNumber="9" type="description">
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<paragraph id="587436C2FFB2FF9FFCE60458F873F964" blockId="8.[825,1475,1258,2010]" pageId="8" pageNumber="9">
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Gene overexpression can be achieved by introducing more copies of the target gene into the MAC in
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<taxonomicName id="9FCB4D41FFB2FF9FFB010478F925FAAD" box="[1246,1368,1328,1352]" class="Hypotrichea" family="Oxytrichidae" genus="Paramecium" kingdom="Chromista" order="Oxytrichida" pageId="8" pageNumber="9" phylum="Ciliophora" rank="species" species="tetraurelia">
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<emphasis id="6ABFEAD0FFB2FF9FFB010478F925FAAD" box="[1246,1368,1328,1352]" italics="true" pageId="8" pageNumber="9">P. tetraurelia</emphasis>
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</taxonomicName>
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(Table 1). A small portion of exogenous DNA introduced into the MAC of
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<taxonomicName id="9FCB4D41FFB2FF9FFC8B0439FFB2FA62" box="[852,975,1391,1415]" class="Hypotrichea" family="Oxytrichidae" genus="Paramecium" kingdom="Chromista" order="Oxytrichida" pageId="8" pageNumber="9" phylum="Ciliophora" rank="species" species="tetraurelia">
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<emphasis id="6ABFEAD0FFB2FF9FFC8B0439FFB2FA62" box="[852,975,1391,1415]" italics="true" pageId="8" pageNumber="9">P. tetraurelia</emphasis>
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</taxonomicName>
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can be integrated into the chromosomes, similar to
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<taxonomicName id="9FCB4D41FFB2FF9FFC8C04C7FFAEFA43" box="[851,979,1422,1446]" class="Oligohymenophorea" family="Tetrahymenidae" genus="Tetrahymena" kingdom="Chromista" order="Hymenostomatida" pageId="8" pageNumber="9" phylum="Ciliophora" rank="genus">
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<emphasis id="6ABFEAD0FFB2FF9FFC8C04C7FFAEFA43" box="[851,979,1422,1446]" italics="true" pageId="8" pageNumber="9">Tetrahymena</emphasis>
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</taxonomicName>
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, while most is added with
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<taxonomicName id="9FCB4D41FFB2FF9FFB3E04C6F926FA43" box="[1249,1371,1423,1446]" class="Hypotrichea" family="Oxytrichidae" genus="Paramecium" kingdom="Chromista" order="Oxytrichida" pageId="8" pageNumber="9" phylum="Ciliophora" rank="genus">
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<emphasis id="6ABFEAD0FFB2FF9FFB3E04C6F926FA43" box="[1249,1371,1423,1446]" italics="true" pageId="8" pageNumber="9">Paramecium</emphasis>
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</taxonomicName>
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telomerelike sequences at both termini and replicates autonomously (
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<bibRefCitation id="3C5A4B33FFB2FF9FFC9C0484FF88FA00" author="Gilley" box="[835,1013,1485,1509]" etAl="et al." firstAuthor="Gilley" pageId="8" pageNumber="9" pagination="4765 - 72" refId="ref9782" refString="Gilley D * Preer JR * Aufderheide KJ et al. Autonomous replication and additionoftelomerelikesequencestoDNAmicroinjectedinto Paramecium tetraurelia macronuclei. Molecular and Cellular Biology 1988; 8: 4765 - 72. hups: // doi. org / 10.1128 / mcb. 8.11.4765 - 4772.1988" type="journal article" year="1988">
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Gilley
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<emphasis id="6ABFEAD0FFB2FF9FFC560487FFC6FA00" box="[905,955,1485,1509]" italics="true" pageId="8" pageNumber="9">et al.</emphasis>
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1988
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</bibRefCitation>
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, Bourgain and Katinka 1991). Not only the linear DNA but also the circular DNA can be maintained by cleavage at non-random sites to convert it from supercoiled to linear (
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<bibRefCitation id="3C5A4B33FFB2FF9FFC5C0762F83AF9A6" author="Godiska" box="[899,1095,1579,1603]" etAl="et al." firstAuthor="Godiska" pageId="8" pageNumber="9" pagination="7590 - 4" refId="ref9827" refString="Godiska R * Aufderheide KJ * Gilley D et al. ºransformation of Paramecium by microinjection of a cloned serotype gene. Proceedings of the National Academy of Sciences of the United States of America 1987; 84: 7590 - 4. hups: // doi. org / 10.1073 / pnas. 84.21.7590" type="journal article" year="1987">
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Godiska
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<emphasis id="6ABFEAD0FFB2FF9FFC000765F873F9A6" box="[991,1038,1579,1603]" italics="true" pageId="8" pageNumber="9">et al.</emphasis>
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1987
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</bibRefCitation>
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,
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<bibRefCitation id="3C5A4B33FFB2FF9FFB8D0762F882F9A6" author="Gilley" box="[1106,1279,1579,1603]" etAl="et al." firstAuthor="Gilley" pageId="8" pageNumber="9" pagination="4765 - 72" refId="ref9782" refString="Gilley D * Preer JR * Aufderheide KJ et al. Autonomous replication and additionoftelomerelikesequencestoDNAmicroinjectedinto Paramecium tetraurelia macronuclei. Molecular and Cellular Biology 1988; 8: 4765 - 72. hups: // doi. org / 10.1128 / mcb. 8.11.4765 - 4772.1988" type="journal article" year="1988">
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Gilley
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<emphasis id="6ABFEAD0FFB2FF9FFB490765F8BBF9A6" box="[1174,1222,1579,1603]" italics="true" pageId="8" pageNumber="9">et al.</emphasis>
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1988
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</bibRefCitation>
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). To guarantee the efficiency of retention of exogenous DNA, linear DNA is used in routine experiments.
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</paragraph>
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<paragraph id="587436C2FFB2FF9EFC8A07C0FE87FEE3" blockId="8.[825,1475,1258,2010]" lastBlockId="9.[113,762,144,262]" lastPageId="9" lastPageNumber="10" pageId="8" pageNumber="9">
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The flanking sequences and target gene are amplified and inserted into the vector (
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<figureCitation id="C0F02A47FFB2FF9FFBF007E1F804F925" box="[1071,1145,1704,1728]" captionStart="Figure 5" captionStartId="8.[129,194,1018,1042]" captionTargetBox="[138,1466,153,980]" captionTargetId="figure-518@8.[128,1475,144,991]" captionTargetPageId="8" captionText="Figure 5. Gene overexpression strategies in ciliates.A* construct of gene overexpression and the homologous recombination in Tetrahymena (a) and positive transformed cell screening (b). Black arrows with coloured tails indicate the primers with 15–20 bp overlap designed for fusion PCR.Red arrows pointing to the plasmid indicate the restriction enzyme digestion sites. B* gene overexpression by injection of more copies in Paramecium. ºelomeres are indicated with red squares at the end of chromosomes. C* gene overexpression by introduction of artificial chromosomes in Oxytricha. Abbreviations:GOI* gene of interest; MAC* macronucleus; MIC* micronucleus; MT1 and MT3* cadmiuminducible metallothionein genes; neo Tet* a synthetic neo gene (neomycin-resistance gene) optimized for codon usage of Tetrahymena; PM* paromomycin." figureDoi="http://doi.org/10.5281/zenodo.14423119" httpUri="https://zenodo.org/record/14423119/files/figure.png" pageId="8" pageNumber="9">Fig. 5B</figureCitation>
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). To meet the requirements of some experiments, a tag sequence can be added immediately after the start codon or before the stop codon of the target gene. The plasmid is linearized by restriction enzyme digestion at the backbone region, then introduced into the MAC of vegetative cells by microinjection. Positive injected cells can be checked using PCR or dot-blot by specifically detecting the DNA that is not present in the genome of
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<taxonomicName id="9FCB4D41FFB2FF9FFB8606CDF8AEF87E" box="[1113,1235,1924,1947]" class="Hypotrichea" family="Oxytrichidae" genus="Paramecium" kingdom="Chromista" order="Oxytrichida" pageId="8" pageNumber="9" phylum="Ciliophora" rank="genus">
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<emphasis id="6ABFEAD0FFB2FF9FFB8606CDF8AEF87E" box="[1113,1235,1924,1947]" italics="true" pageId="8" pageNumber="9">Paramecium</emphasis>
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</taxonomicName>
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. Owing to the high concentration of DNA microinjected, the target gene in the transformed cells is prominently overexpressed compared with the wild-type cells. Moreover, the injected linear constructs can be maintained for a long time in vegetative cells without decreasing rapidly during cell division (
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<bibRefCitation id="3C5A4B33FFB3FF9EFE490186FE3EFF02" author="Gilley" box="[406,579,207,231]" etAl="et al." firstAuthor="Gilley" pageId="9" pageNumber="10" pagination="4765 - 72" refId="ref9782" refString="Gilley D * Preer JR * Aufderheide KJ et al. Autonomous replication and additionoftelomerelikesequencestoDNAmicroinjectedinto Paramecium tetraurelia macronuclei. Molecular and Cellular Biology 1988; 8: 4765 - 72. hups: // doi. org / 10.1128 / mcb. 8.11.4765 - 4772.1988" type="journal article" year="1988">
|
||
Gilley
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||
<emphasis id="6ABFEAD0FFB3FF9EFE050186FE77FF02" box="[474,522,207,231]" italics="true" pageId="9" pageNumber="10">et al.</emphasis>
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||
1988
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</bibRefCitation>
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||
). However, DNA is lost with the degradation of the old MAC during conjugation.
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</paragraph>
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</subSubSection>
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</treatment>
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