Gene analysis techniques最新文献_第8页

Detection of recombination events in human cells using cloned cytomegalovirus DNA fragments as probes 克隆巨细胞病毒DNA片段作为探针检测人细胞重组事件

Gene analysis techniques Pub Date : 1988-07-01 DOI: 10.1016/0735-0651(88)90009-X

Claude Hamelin , Jocelyn Yelle , Michel Dion

引用次数: 1

Differential behavior of liposome-introduced specific RNAs in living Drosophila cells 脂质体引入的特异性rna在果蝇细胞中的差异行为

Gene analysis techniques Pub Date : 1988-07-01 DOI: 10.1016/0735-0651(88)90006-4

Robert H. Gross , Dianne L. Rosen , Martin S. Cetron

{"title":"Differential behavior of liposome-introduced specific RNAs in living Drosophila cells","authors":"Robert H. Gross , Dianne L. Rosen , Martin S. Cetron","doi":"10.1016/0735-0651(88)90006-4","DOIUrl":"10.1016/0735-0651(88)90006-4","url":null,"abstract":"<div><p>We have developed a protocol for efficiently introducing macromolecules into <em>Drosophila</em> tissue culture cells using liposomes. By carefully adjusting the fusion parameters, conditions have been established to routinely encapsulate 15–30% of the starting material into liposomes and to introduce 20–30% of the liposome-encapsulated material into the cells during a 30-minute fusion period. Essentially, all of the cells receive material from the liposomes and 10<sup>9</sup> cells can be fused at once. The fusion does not have any measureable effect on cell viability as assayed by trypan blue exclusion, growth rate, and cell morphology. We have utilized this technique to introduce radioactive RNAs into nonradioactive cells, thus enabling the behavior of the introduced RNAs to be followed unambiguously. Liposome-introduced small nuclear RNAs (snRNAs) are stable in the cell for at least 25 hours (approximately two cell generations), with 80% of the radioactivity remaining trichloroacetic acid (TCA) precipitable and the gel electrophoresis pattern remaining essentially unchanged. This is in contrast to liposome-introduced cytoplasmic RNAs, which are only 20% TCA precipitable after the first hour. In the cell, the introduced snRNAs attain a 10–35-fold higher concentration in the nucleus than the cytoplasm. Nuclear accumulation is not seen with <em>Drosophila</em> tRNA or 5s RNA, both of which attain the same nuclear as cytoplasmic RNA concentration.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"5 4","pages":"Pages 63-72"},"PeriodicalIF":0.0,"publicationDate":"1988-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(88)90006-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13607373","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}

引用次数: 2

Antibodies introduced into living cells with liposomes localize specifically and inhibit specific intracellular processes 通过脂质体引入活细胞的抗体可以特异地定位并抑制特定的细胞内过程

Gene analysis techniques Pub Date : 1988-07-01 DOI: 10.1016/0735-0651(88)90007-6

W. Scott Thompson, Robert H. Gross

{"title":"Antibodies introduced into living cells with liposomes localize specifically and inhibit specific intracellular processes","authors":"W. Scott Thompson, Robert H. Gross","doi":"10.1016/0735-0651(88)90007-6","DOIUrl":"10.1016/0735-0651(88)90007-6","url":null,"abstract":"<div><p>We have developed a system for efficiently packaging antibodies and other macromolecules into lipsomes and then delivering the encapsulated molecules into living cells through liposome-cell fusion. Fusion is very efficient, and all cells can be demonstrated to contain liposome-delivered antibodies by staining by staining with a fluorescent second antibody. Using lupus antibodies directed against small nuclear ribonucleoprotein components of the cell, we were able to demonstrate strong nuclear localization, while control antibodies showed a general diffuse distribution throughout the cell. Lupus antibodies directed against ribosomes, on the other hand, strongly localized in the nucleolus and the cytoplasm with very little nucleoplasmic localization. Antitubulin antibodies predominantly localized in the cytoplasm. These results show that antibodies can survive liposome packaging and can retain their ability to recognize and bind to their specific antigens in the living cell. It also indicates that the nuclear envelope does not present a barrier to the liposome-introduced antibodies in <em>Drosophila</em> tissue culture cells. To determine if the antibodies were capable of interfering with cellular processes in vivo, we measured the effects of liposome-introduced antiribosome antibodies on translation and antitubulin antibodies on mitosis. In both cases, there was a significant inhibition suggesting that the antibodies can be used to interfere with specific functions at specific times in vivo.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"5 4","pages":"Pages 73-79"},"PeriodicalIF":0.0,"publicationDate":"1988-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(88)90007-6","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14321385","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}

引用次数: 9

Molecular pathogenesis of human immunodeficiency virus infection 人类免疫缺陷病毒感染的分子发病机制

Gene analysis techniques Pub Date : 1988-05-01 DOI: 10.1016/0735-0651(88)90015-5

Arnold B. Rabson , Scott Koenig , Daryl F. Daugherty , Howard E. Gendelman

{"title":"Molecular pathogenesis of human immunodeficiency virus infection","authors":"Arnold B. Rabson , Scott Koenig , Daryl F. Daugherty , Howard E. Gendelman","doi":"10.1016/0735-0651(88)90015-5","DOIUrl":"10.1016/0735-0651(88)90015-5","url":null,"abstract":"<div><p>Molecular studies of the pathogenesis of human immunodeficiency virus (HIV) infections have proceded rapidly following the molecular cloning and nucleotide sequence analysis of the HIV genome. Correlation of biochemical and functional studies of HIV-infected cells with the HIV nucleotide sequence has allowed the identification and preliminary functional characterization of many HIV proteins. These include structural proteins (<em>gag</em>), viral enzymes (<em>pol</em>), and viral regulatory proteins (<em>tat, art</em>). Cloned HIV DNA segments have been utilized as probes for in situ nucleic acid hybridization to study the distribution of HIV-infected cells in acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC) patients. These studies have demonstrated the infection of macrophages as an important component of HIV-induced neurologic disease. Only very low numbers of HIV-infected lymphocytes can be identified in the peripheral blood of infected individuals. Thus, the mechanism of CD4 cell depletion in the pathogenesis of AIDS remain obscure.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"5 3","pages":"Pages 41-53"},"PeriodicalIF":0.0,"publicationDate":"1988-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(88)90015-5","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14189300","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}

引用次数: 4

An improved procedure for measuring DNA replication with transient assays in eukaryotic cells 真核细胞瞬时测定DNA复制的改进方法

Gene analysis techniques Pub Date : 1988-05-01 DOI: 10.1016/0735-0651(88)90016-7

Maggie Sullivan, William R. Folk

引用次数: 1

Synthesis of long cDNA from viral RNA template 从病毒RNA模板合成长cDNA

Gene analysis techniques Pub Date : 1988-05-01 DOI: 10.1016/0735-0651(88)90017-9

J.A. Lenstra, R.J. De Groot, L. Jacobs, J.G. Kusters, H.G.M. Niesters, B.A.M. Van Der Zeijst

引用次数: 6

A simple method of examining retroviral species produced by infected cells 一种检测受感染细胞产生的逆转录病毒种类的简单方法

Gene analysis techniques Pub Date : 1988-03-01 DOI: 10.1016/0735-0651(88)90022-2

Laura S. Levy, Patricia A. Lobelle-Rich

引用次数: 1

A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing 支持高效转录和前mrna剪接的核提取物制备的小规模程序

Gene analysis techniques Pub Date : 1988-03-01 DOI: 10.1016/0735-0651(88)90023-4

Kevin A.W. Lee, Albrecht Bindereif, Michael R. Green

{"title":"A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing","authors":"Kevin A.W. Lee, Albrecht Bindereif, Michael R. Green","doi":"10.1016/0735-0651(88)90023-4","DOIUrl":"10.1016/0735-0651(88)90023-4","url":null,"abstract":"<div><p>A convenient and rapid method for preparing soluble extracts from the nuclei of as few as 3 × 10<sup>7</sup> mammalian cells (miniextract procedure) is described. By several criteria, miniextracts are comparable to nuclear extracts prepared from large numbers of cells by the conventional procedure. Miniextracts are able to support efficient transcription of a variety of class II promoters. In addition, DNase I footprinting and gel retardation assays can be performed directly in miniextracts, enabling the detection of sequence-specific DNA-binding proteins. Besides transcription, miniextracts efficiently carry out pre-mRNA splicing and allow formation and fractionation of previously characterized splicing complexes. The small-scale procedure enables simultaneous preparation of multiple extracts from a variety of cell types under different experimental conditions. Moreover, the use of small amounts of cells allows minimal expenditure of valuable or expensive materials such as radioactive compounds. Consequently, the procedure is highly advantageous for biochemical analysis of transcription and RNA processing in mammalian cells.</p></div>","PeriodicalId":77714,"journal":{"name":"Gene analysis techniques","volume":"5 2","pages":"Pages 22-31"},"PeriodicalIF":0.0,"publicationDate":"1988-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0735-0651(88)90023-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14321383","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}

引用次数: 432

Optimal conditions for supercoil DNA sequencing with the Escherichia coli DNA polymerase I large fragment 大肠杆菌DNA聚合酶I大片段超螺旋DNA测序的最佳条件

Gene analysis techniques Pub Date : 1988-03-01 DOI: 10.1016/0735-0651(88)90024-6

Heon M. Lim, Jacques J. Pène

引用次数: 53

In vivo plasmid construction by integrative recombination within a 156 bp sequence homology 在156bp序列同源性内整合重组的体内质粒构建

Gene analysis techniques Pub Date : 1988-01-01 DOI: 10.1016/0735-0651(88)90019-2

Simon F. Park, Gordon S.A.B. Stewart

引用次数: 2