[Isolation of fragments of native DNA containing structural genes at the end].
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
G P Georgiev
Publications and source records attributed to G P Georgiev.
The existence of triphosphorylated 5'-ends in virus-specific RNA isolated from SV-40 transformed cells.
The question about the nature of promoters in the transcriptional units containing SV-40 sequences in transformed cells was analyzed. It was found that the pulse-labeled RNA hybridizing to SV-40 DNA contains small but significant amounts of triphosphorylated 5'-ends detected as pppGp in alkaline hydrolyzates of this RNA. In another series of experiments the fragments of RNA containing triphosphorylated 5'-ends about 100 nucleotides in length have been isolated by hydroxyapatite chromatography. Some of them form hybrids with SV-40 DNA. The conclusion is drawn that at least some of SV-40 promoters are used for transcription initiation in SV-40 transformed cells.
Alkaline Phosphatase↗
On the nature of 5' termini in nuclear pre-mRNA of Ehrlich carcinoma cells.
5' terminal nucleosides of nuclear pre-mRNA of Ehrlich ascites carcinoma cells were analyzed by a combination of different chromatographic methods and phosphatase treatment. The heavy nuclear pre-mRNA contains mainly unblocked triphosphorylated nucleosides at the 5' end, although some capped 5' ends could also be found. In this respect, it differs from cytoplasmic poly(A)+ mRNA which contains blocked 5' termini and no triphosphorylated ends. The 5' terminal nucleotides in pre-mRNA are pppGp and pppAp (in a ratio of 3:2). The determination of pppNp content in poly (A)+, poly(U)+, and poly (A)-(U)- fragments of RNA has been used as an approach to establish the topography of pre-mRNA. We also established that the technique for isolation of triphosphorylated 5' terminal fragments of RNA based on hydroxyapatite chromatography (Bajszár, Samarina, and Georgiev, 1974) is still valid in the presence of blocked oligonucleotides. The latter do not interfere with fragments containing free triphosphate groups. Using this technique, we showed that a small but significant portion of triphosphorylated 5' end fragments of 100 nucleotides in length contain oligo(U) sequences reacting with poly(A)-Sepharose.
Base Sequence↗
Minichromosome of simian virus 40: presence of histone HI.
In contrast to conclusions of previous studies /I-3/ claiming the absence of histone HI from the SV40 and polyoma viral minichromosomes we have found that a preparation of purified SV40 minichromosomes does contain histone HI. The content of HI in relation to other four histones in the SV40 minichromosomes is close to that in the cellular chromatin. Histone HI in the isolated SV40 minichromosomes is bound apparently to internucleosomal DNA stretches as was shown already for HI in the cellular chromatin /4/. In addition it was found that more than 90% of the purified SV40 minichromosomes migrated as a single discrete deoxyribonucleoprotein band upon agarose gel electrophoresis.
Cell Fractionation↗
DNA-synthesis on giant nuclear RNA by AMV DNA polymerase.
The reverse transcription of pre-mRNA isolated from rat liver or mouse Ehrlich ascites carcinoma cells with the aid of hot phenol fractionation technique is described. Pre-mRNA isolated at 85 degrees C is a more active template than the 65 degree C fraction. The addition of oligo(dT) as a primer strongly stimulated the template activity of the 65 degree C fraction. The size of product corresponds to a sedimentation value of 7 S as measured in alkaline sucrose gradient and is essentially less than the size of template.
Animals↗
[Selection and some properties of recombinant clones of lambda bacteriophage containing structural genes of Drosophila melanogaster].
The lambdagt clones containing fragments of D. melanogaster genome were prepared and characterized by hybridization of their DNA with complementary RNA synthesized on the lambdagt DNA, C fragment of lambdagt DNA and total D. melanogaster DNA, and also with mRNA and a stable cytoplasmic poly(A)-RNA both isolated from D. melanogaster cells grown in culture. The technique for a simple selection of hybrid clones is described. The hybridization with mRNA allows one to select the clones containing structural genes of D. melanogaster. It was found that in all cases when the clone contains the structural gene it also contains the reiterated base sequences of the D. melanogaster genome. Several clones containing D. melanogaster DNA fragments with a size of (2--4) X 10(6) daltons which hybridized with a relatively large portion of mRNA were selcted for further analysis.
Animals↗
A new type of organization of the genetic material in eukaryotes.
An investigation of the properties of a number of genes of Drosophila, obtained by cloning recombinant DNAs, led to the detection of a new type of organization of genetic material. It was found that a number of actively working structural genes of Drosophila are represented by a large number of copies, scattered over its chromosomes. Their localization in the chromosomes is variable, although they are always detected in regions of intercalary heterochromatin. The latter evidently is a site of accumulation of various multiple genes.
Alleles↗
[Isolation of DNA fraction bound to the axial structure of metaphase chromosomes and studies of renaturation].
Mitotic chromosomes of L cells (metaphase plates) were dehistonized by centrifugation through a layer of 2 M NaCl and then treated with restriction endonuclease EcoRI and HindIII. Alternatively, they were pretreated with EcoRI endonuclease. The DNA remaining attached to the axial structure of the chromosomes was isolated and investigated in renaturation experiments. It was found to be enriched in reiterated base sequences belonging to the satellite and to abundant intermediate repeats.
Animals↗
[Structure of nuclear pre-mRNA. XI. Triphosphorylated and blocked 5'-ends in the pre-mRNA].
The nature of the 5'-termini in pre-mRNA isolated from Ehrlich carcinoma cells has been investigated. To discriminate between triphosphorylated 5'-ends and capped structures different methods were used including treatment by alkaline phosphatase and several chromatographic methods. It was shown that heavey pre-mRNA contains a significant number of non-blocked triphosphorylated nucleotides at the 5'-end termini. However, phosphatase resistent, blocked 5'-termini were also found. 5'-terminal nucleotides in triphosphorylated pre-mRNA are G in a 3 : 2 ratio. In contrast to nuclear pre-mRNA cytoplasmic poly(A)+mRNA does not contain triphosphorylated 5'-ends but does contain the "cap" structure only. To elucidate the pre-mRNA topography the localization of homopolymeric regions of pre-mRNA, poly(A) and oligo(U), in relation to 5'terminal structures has been investigated. The experiments showed that the distance between 3'-terminal poly(A) sequences and 5'-end triphosphates is longer than 1500--2000 nucleotides. At the same time the distance between the latter and oligo(U) in pre-mRNA is much shorter.
Alkaline Phosphatase↗
[DNA synthesis on the heterogeneous nuclear RNA template catalysed by DNA polymerase of avian myeloblastosis virus].
Template activity of nuclear pre-mRNA has been investigated in DNA-polymerase reaction. Active synthesis of DNA was demonstrated on pre-mRNA as a template in the absence of primer. A part of synthetic activity may be attributed to the traces of DNA present in the pre-mRNA preparation. Addition of oligo(dT)10 to the template stimulated the synthesis of DNA product due to transcription of heteropolymeric regions near the poly(A). The rate of DNA synthesis was different depending on the fraction of template used: the RNA extracted by hot phenol at 85 degrees showed higher template activity without adding of primer than the 65 degrees C fraction. On the contrary 65 degrees C pre-mRNA which is known to contain greater quantity of molecules with poly(A) at the 3'-end is more strongly stimulated by addition of oligo(dT). The nuclear RNA corresponding to the precursors of rRNAs extracted at 40 degrees C were not transcribed by the reverse transcriptase. The size of the DNA-product (about 7-8S in alkaline sucrose gradient) did not depend on the size of the template neither on the presence of oligo(dT)10 primer. The inhibition of the second DNA strand synthesis with actinomycin D had also no influence on the size of DNA-product.
Avian Leukosis Virus↗