Gene analysis techniques最新文献

{"title":"Fragment tagging","authors":"Jill H Zeilstra-Ryalls ,&nbsp;Ronald L Somerville","doi":"10.1016/0735-0651(90)90029-F","DOIUrl":"10.1016/0735-0651(90)90029-F","url":null,"abstract":"<div><p>A tactic known as fragment tagging, which has proven to be exceptionally useful in expediting DNA cloning and plasmid construction schemes, is described. The advantage of fragment tagging is that it facilitates the isolation of specific plasmid DNA molecules present in small amounts within mixed pools of DNA. Four examples that illustrates several variations of the fragment tagging concept are presented.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"7 6","pages":"Pages 151-159"},"PeriodicalIF":0.0,"publicationDate":"1990-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(90)90029-F","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13232246","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: Implications for glycosylation and CD4 binding","authors":"Cheryl Isaac Murphy,&nbsp;Michael Lennick,&nbsp;Sophie M Lehar,&nbsp;Gerald A Beltz,&nbsp;Elihu Young","doi":"10.1016/0735-0651(90)90030-J","DOIUrl":"10.1016/0735-0651(90)90030-J","url":null,"abstract":"<div><p>Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in <em>Spodoptera frugiperda</em> (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160Δ (full length gp160 minus the transmembrane and cytoplasmic region of gp41) were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a contransfection with DNA from <em>Autographa californica</em> nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycoslated and nonglycosylated versions of the HIV proteins as determined by ³H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160Δ specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160Δ proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4. We conclude that production of native HIV envelope proteins, as measured by addition of carbohydrate side chains and ability to bind CD4, peaks early after infection in baculovirus-infected insect cells.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"7 6","pages":"Pages 160-171"},"PeriodicalIF":0.0,"publicationDate":"1990-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(90)90030-J","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13232247","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"GATA's new format","authors":"Cassandra L Smith (Executive Editor)","doi":"10.1016/0735-0651(90)90012-5","DOIUrl":"https://doi.org/10.1016/0735-0651(90)90012-5","url":null,"abstract":"","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"7 5","pages":"Page 93"},"PeriodicalIF":0.0,"publicationDate":"1990-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(90)90012-5","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"71868093","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Alternatives to YACs","authors":"Nat L Sternberg","doi":"10.1016/0735-0651(90)90018-B","DOIUrl":"10.1016/0735-0651(90)90018-B","url":null,"abstract":"","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"7 5","pages":"Pages 126-132"},"PeriodicalIF":0.0,"publicationDate":"1990-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(90)90018-B","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13247322","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Generating highly labeled oligonucleotides for DNA-protein interaction","authors":"Steven Dooley,&nbsp;Cornelius Welter,&nbsp;Birgit Theisinger,&nbsp;Nikolaus Blin","doi":"10.1016/0735-0651(90)90019-C","DOIUrl":"10.1016/0735-0651(90)90019-C","url":null,"abstract":"<div><p>We developed a new strategy to prepare double-stranded oligonucleotides containing recognition sites for specific binding proteins to examine DNA-protein interactions in various assays (gel mobility shift, UV-crosslinking, and affinity chromatography). The advantages of our procedure are as follows. Only one strand needs to be synthesized using a commercial oligonucleotide synthesizer. The probes can be labeled to a high specific activity and the exact position of labeling can be chosen, which is necessary for UV-crosslink studies. Furthermore, multimeric binding sites for efficient DNA affinity chromatography can easily be generated. It is also possible to precisely place modified bases without the need for chemical precursors. Using this protocol, more detailed information about the binding protein factors and their behavior in interaction with recognition sites can be obtained.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"7 5","pages":"Pages 133-137"},"PeriodicalIF":0.0,"publicationDate":"1990-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(90)90019-C","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13247324","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Direct sequencing of PCR products using the Maxam-Gilbert method","authors":"Stefan Stamm ,&nbsp;Frank M. Longo","doi":"10.1016/0735-0651(90)90021-7","DOIUrl":"10.1016/0735-0651(90)90021-7","url":null,"abstract":"<div><p>Direct sequencing of polymerase chain reaction (PCR) products by using the Maxam-Gilbert method is described. In this method, one of the primers is end labeled. Thus it is possible to sequence the reaction product directly following purification using this chemical method.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"7 5","pages":"Pages 142-143"},"PeriodicalIF":0.0,"publicationDate":"1990-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(90)90021-7","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13247325","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"YAC cloning of telomeres","authors":"Jan-Fang Cheng ,&nbsp;Cassandra L Smith","doi":"10.1016/0735-0651(90)90017-A","DOIUrl":"10.1016/0735-0651(90)90017-A","url":null,"abstract":"<div><p>Human telomeres have been succesfully cloned in <em>Saccharomyces cerevisiae</em> by complementing deficient yeast artificial chromosomes (YACs). This technique allows cloning of DNA sequences that can recognize particular chromosomal ends, and therefore facilitates the mapping of eukaryotic genomes. Although the biology of adopting foreign telomeres in yeast is not fully understood, the cloning system itself seems to be a useful tool for constructing telomeric DNA libraries from higher eukaryotes. Here we describe the techniques that are currently being used in cloning of telomeric DNA.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"7 5","pages":"Pages 119-125"},"PeriodicalIF":0.0,"publicationDate":"1990-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(90)90017-A","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13288568","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"YAC cloning of DNA embedded in an agarose matrix","authors":"Mary Kay McCormick ,&nbsp;Stylianos E Antonarakis ,&nbsp;Philip Hieter","doi":"10.1016/0735-0651(90)90016-9","DOIUrl":"10.1016/0735-0651(90)90016-9","url":null,"abstract":"<div><p>Yeast artificial chromosome (YAC) cloning of DNA in agarose is an alternative method to cloning from aqueous solutions. It minimizes any shearing that may result from handling of high molecular weight DNA and can be done with nanogram to microgram amounts of material, which facilitates construction of YACs from sources of DNA other than genomic DNA isolated from cells. The average size of the YACs recovered (200–1000 kb) and efficiency of transformation of ligation products (200–1000 cfu/μg) are similar to those reported using aqueous protocols. This method has been used to construct chromosome specific YACs, and it should be possible to apply the technique to the construction of chromosome specific libraries using flow sorted chromosomes as source material, and the cloning of restriction fragments isolated by preparative pulsed field gel electrophoresis.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"7 5","pages":"Pages 114-118"},"PeriodicalIF":0.0,"publicationDate":"1990-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(90)90016-9","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13247321","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"YAC cloning: Options and problems","authors":"David T Burke","doi":"10.1016/0735-0651(90)90013-6","DOIUrl":"10.1016/0735-0651(90)90013-6","url":null,"abstract":"","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"7 5","pages":"Pages 94-99"},"PeriodicalIF":0.0,"publicationDate":"1990-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(90)90013-6","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13247326","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Current methods for YAC clone characterization","authors":"David L. Nelson","doi":"10.1016/0735-0651(90)90014-7","DOIUrl":"10.1016/0735-0651(90)90014-7","url":null,"abstract":"<div><p>Yeast artificial chromosome (YAC) cloning has allowed isolation of much longer DNA fragments than was previously possible. While this technology makes feasible the isolation of large regions of complex genomes in overlapping cloned segments, it has also required altered methods for manipulation of cloned DNAs. In particular, the isolation of insert sequences from YAC clones has been especially difficult. Methods for characterization of YAC inserts are described and compared.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"7 5","pages":"Pages 100-106"},"PeriodicalIF":0.0,"publicationDate":"1990-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(90)90014-7","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13247397","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}