ABSTRACT
The conditions for hybridization and detection of enzyme-labeled probes have been optimized in our laboratory for use with oligonucleotides coupled to alkaline phosphatase. We have examined several enzyme-linked probe which are complimentary to commonly used variable number of tandem repeats (VNTR) loci to determine the feasibility of using chemiluminescence for routine application in forensic DNA analysis. It was found that a chemiluminescent detection system employing an alkaline phosphatase activated dioxetane in the presence of chemiluminescent enhancers provides a high degree of sensitivity in hybridization protocols with a significant savings in overall filter processing time. The chemiluminescent system achieved equal or greater sensitivity that observed for 32P-labeled probes in much shorter development times. Furthermore, a new chemiluminescent substrate, Lumi-Phos Plus, has recently been investigated and found to further decrease the filter development time for forensic assays.
Subject(s)
DNA Probes/chemistry , DNA/analysis , Forensic Medicine/methods , Indicators and Reagents/chemistry , Luminescent Measurements , Alkaline Phosphatase , Alleles , Humans , Immunoblotting , Organic ChemicalsABSTRACT
The amounts of mouse beta major-globin mRNA and globin gene upstream RNA polymerase III transcripts were compared during the differentiation of purified erythroid colony-forming progenitor cells (CFU-E) in vitro. The accumulation of each RNA was determined relative to the 100% levels in fetal liver erythroid cells. The mRNA level was low in freshly isolated CFU-E and began to accumulate only after a lag period of approximately 2-4 h. It continued to accumulate for approximately 40 h thereafter, at which point it was comparable to that in the fetal liver. In contrast, in three of four CFU-E preparations, the relative level of upstream RNAs was high in freshly isolated CFU-E and reached the maximal level (defined as 100% of the fetal liver level) more rapidly than did the mRNA. The early induction and accelerated accumulation of upstream RNAs in immature erythroid cells suggest some role for these RNAs at an early stage of globin gene activation.
Subject(s)
Erythrocytes/cytology , Genes , Globins/genetics , Hematopoietic Stem Cells/cytology , Animals , Cells, Cultured , Erythrocytes/metabolism , Hematopoietic Stem Cells/metabolism , Mice , RNA/metabolism , RNA, Messenger/metabolism , Spleen/cytology , Spleen/metabolism , Transcription, GeneticABSTRACT
A base substitution in the 5'-flanking region of a human fetal globin gene is associated with abnormal fetal hemoglobin production. It also reduces by 5- to 10-fold in vitro transcription of the gene by RNA polymerase III. We discuss potential links between polymerase III transcription and abnormal hemoglobin production.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , Fetal Hemoglobin/genetics , Genes , Globins/genetics , Mutation , RNA Polymerase III/metabolism , Transcription, Genetic , Amanitins/pharmacology , Base Sequence , Genes/drug effects , HumansABSTRACT
The initiation of transcription by RNA polymerase II in isolated murine erythroleukemia cell nuclei was investigated by isolating newly synthesized gamma-thio (gamma-S-)-triphosphate-labeled transcripts by Hg-agarose chromatography. The 5' terminus of transcripts initiated in vitro with [gamma-35S]ATP or [gamma-35S]GTP was identified as the thiotetraphosphate in alkaline hydrolysis products from Hg-agarose-selected RNA. Additional control experiments analyzing the nuclear transcription of two well characterized tRNA genes showed that each gene was initiated with the proper triphosphate, either gamma-S-ATP or gamma-S-GTP, indicating little, if any, exchange of the gamma-S-labeled substrate to the other triphosphates. As determined by S1 mapping, newly synthesized beta-globin gene transcripts initiate only with gamma-S-ATP. Their 5'-terminus is located at the cap site, and their synthesis is inhibited by 1 microgram alpha-amanitin/ml. In reactions containing gamma-S-ATP but not gamma-S-GTP, several additional initiation sites are observed that are located in the 5'-flanking region. We conclude that RNA polymerase II can initiate transcription at the cap site in isolated nuclei.
Subject(s)
Globins/biosynthesis , Transcription, Genetic , Amanitins/pharmacology , Animals , Cell Line , Friend murine leukemia virus , Globins/genetics , Leukemia, Erythroblastic, Acute , Mice , RNA Caps , RNA Polymerase II/metabolism , RNA, Transfer/biosynthesis , Thionucleotides/metabolism , Transcription, Genetic/drug effectsABSTRACT
A small proportion of the RNAs of mouse reticulocytes consists of beta major-globin mRNA sequences linked to sequences transcribed from the 5'-flanking region of the beta major-globin gene. These upstream RNAs are polyadenylylated and contain 700-800 nucleotides, and their 5' regions are heterogeneous. RNAs with similar or identical 5' regions are transcribed in cell-free extracts from a circular mouse beta major-globin gene template. Synthesis of most of the upstream RNAs in vitro is not inhibited by low levels (1 microgram/ml) of alpha-amanitin, indicating that they are transcribed by an enzyme(s) different from RNA polymerase II. During culture of mouse erythroleukemia cells with dimethyl sulfoxide, globin mRNA and upstream RNAs accumulate with similar kinetics. In contrast, upstream RNAs are not detected in hemin-treated cells.
Subject(s)
Amanitins/pharmacology , Genes , Globins/genetics , RNA, Messenger/genetics , Transcription, Genetic/drug effects , Animals , Base Sequence , Cell Line , HeLa Cells/metabolism , Humans , Leukemia, Erythroblastic, Acute/metabolism , Leukemia, Experimental/metabolism , Mice , Molecular Weight , PlasmidsABSTRACT
We have investigated the intracellular location of RNAs transcribed from transfected DNA. COS cells transfected with a clone containing the human adult beta globin gene contain three classes of globin RNAs. Their 3' termini and splice sites are indistinguishable from those of mature reticulocyte beta globin mRNA, and they are polyadenylated. However, as determined by S1 mapping, their 5' sequences are different. The 5' terminus of one is the same as that of mature beta globin mRNA (+1, cap site). The presumed 5' terminus of the second is located 30 nucleotides downstream from the cap site (+30). The third class contains additional nucleotides transcribed from sequences located 5' to the cap site (5' upstream RNA). The 5' upstream RNA molecules are restricted to the nucleus and are more stable than heterogeneous nuclear RNA. The +30 and +1 RNAs are located primarily in the cytoplasm. The data support the notion that nucleotide sequences and/or secondary modifications in the 5' region determine if an RNA is to be transported.
Subject(s)
Genes , Globins/genetics , RNA, Messenger/analysis , RNA/analysis , Transfection , Animals , Cell Line , Chlorocebus aethiops , Cloning, Molecular , Haplorhini , Humans , Nucleic Acid Hybridization , Plasmids , Reticulocytes/analysis , Subcellular FractionsABSTRACT
We have investigated low abundance RNAs transcribed in vitro and in vivo from the human beta-globin gene. These RNAs contain globin mRNA sequences covalently linked to sequences transcribed from the 5' flanking region between -235 and the mRNA cap site (+1). Their synthesis in vitro is sensitive to high (100 micrograms/ml) levels of alpha-amanitin but not to low (2 micrograms/ml) levels, and one region of the DNA template bordering their 5' termini is similar to a small segment of Alu repetitive DNA and to the RNA polymerase III promoter consensus sequence. Therefore, these RNAs are transcribed by RNA polymerase III but extend into the mRNA-coding region that is usually transcribed by polymerase II. The polymerase III transcripts are polyadenylated and are probably spliced. Their presence in bone marrow cells and peripheral blood reticulocytes implies that they play some role in the erythroid cell.
