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Biomedical subjects

G P Georgiev

Publications and source records attributed to G P Georgiev.

At least 19 recordsLinked to original sources
PubMed1979

Hybridization properties of sequences adjacent to triphosphorylated 5'-ends of nuclear pre-mRNA from mouse Ehrlich carcinoma.

Triphosphorylated 5'-end fragments about 100 nucleotides long were prepared from purified nuclear pre-mRNA using a modified hydroxyapatite method /1/. These fragments as well as fragments of total pre-mRNA of the same size were polyadenylated in vitro by ATP:RNA adenyltransferase and used as templates for the synthesis of [32P] cDNA by reverse transcriptase in the presence of an oligo(dT) primer. The use of cDNA transcribed from the triphosphorylated 5'-end fragments of pre-mRNA (5'-cDNA) and from the total pre-mRNA fragments allows one to calculate the complexity of the 5'-end fraction pre-mRNA and to detect these sequences in polysomal mRNA. Sequences adjacent to 5'-phosphorylated ends of pre-mRNA represent a specific class of sequences with a complexity of about 200 kb. It was also found that about 25% of total pre-mRNA and about a half of sequences adjacent to triphosphorylated 5'-ends are present in polysomal mRNA. A high homology between triphosphorylated 5'-end fragments of pre-mRNA and mRNA sequences may be explained in terms of splicing. Less than 30% of 5'-cDNA hybridized to moderately repetitive DNA while most of them are represented by unique DNA sequences. About 15% of 5'-cDNA contained oligo(dA) sequences originated from oligo(U) in pre-mRNA from which it was transcribed.

Animals↗
PubMed1979

Long double-stranded sequences (dsRNA-B) of nuclear pre-mRNA consist of a few highly abundant classes of sequences: evidence from DNA cloning experiments.

DNA preparations from about hundred randomly selected clones containing mouse DNA fragments were screened for the existence of sequences complementary to long double-stranded regions of pre-mRNA able to snap back after melting (dsRNA-B). Many clones containing such sequences were found. The cloned sequences can be subdivided into three groups: (1) those complementary to about a half (at least to 30-40%) of the total dsRNA, designated as sequences B1; (2) those complementary to a part of sequence B1; and (3) sequences complementary to about a quarter (at least to 15%) of the total dsRNA referred to as sequence B2. The size of DNA sequence complementary to dsRNA is about 400 base pairs. Melting experiments with hybrids show that the members of B1 family are very similar if not identical, while the divergence among B2 sequences is higher, but still the number of substitutions does not exceed 9% of bases. Thus, the major part of dsRNA-B consists of a small number of highly abundant sequences as was suggested earlier on the basis of renaturation kinetics /1-3/. Sequences B1 and B2 are represented by many copies in the mouse genome and in pre-mRNA, and many of them probably do not form hairpin-like structures.

Animals↗
PubMed1978

Reiterated genes with varying location in intercalary heterochromatin regions of Drosophila melanogaster polytene chromosomes.

The localization of two cloned D. melanogaster DNA fragments in polytene chromosomes was determined by means of in situ hybridization. These different fragments (Dm 225 and Dm 234B) are present in the genome in hundreds copies and contain genes whose transcription yields two different classes in abundant mRNA (Ilyin et al., 1976, 1977; Tchurikov et al., 1978). About 20--30 sites of these genes are demonstrable in the polytene chromosomes of a given stock. There are small but significant variations in the number and localization of these sites among individuals of the same stock. On the other hand, different stocks of D. melanogaster have an utterly different distribution of revealed hybridization sites in the polytene chromosomes. The location of both fragments (Dm 225 and Dm 234) was found to be virtually identical within any given stock of D. melanogaster. 69 sites for localization of Dm 225 or Dm 234 genes were detected in the chromosomes of 11 individuals studied. At least 50 (and up to 62) of them coincide with intercalary heterochromatin regions which are known to be characterized by ectopic pairing, late replication and the presence of "weak spots" in the chromosome. The ability of Dm225 and Dm 234 to code for the "abundant" classes of messenger RNA (Ilyin et al., 1976) and the fact that their location may coincide with the histone and ribosomal genes suggest that intercalary heterochromatin regions are "nests" containing various types of actively transcribable tandem-repeated genes coding for common "household" cell functions.

Animals↗
PubMed1978

The properties of gene Dm 225, a representative of dispersed repetitive genes in Drosophila melanogaster.

The properties of Dm 225 DNA, a fragment of D.melanogaster genome 2.9 kb in length excised by EcoRI endonuclease and cloned in the lambda gt phage or pMB9 plasmid, are described. The DNA hybridizes to a significant portion (0.8%) of total polysomal poly(A)(+)RNA (mRNA). The size of the hybridizing mRNA is about 2.3 kb (19S); it is present in the fraction of heavy polysomes. Dm 225 DNA fragments obtained with the aid of Hae III endonuclease have been mapped. mRNA hybridizes with all the fragments. In one of the end fragments, the 3'-end of mRNA has been localized and thus the direction of transcription determined. About 250 copies of the gene Dm 225 are present in the haploid genome of D.melanogaster, and all of them have the same size upon restriction with EcoRI endonuclease. On the other hand, the sequences of the genome adjacent to Dm 225 DNA are different and may vary from one cell line to another as evidenced by experiments in which the D.melanogaster DNA was restricted by Hind III endonuclease. In combination with in situ hybridization data /1,2/ the results obtained in this paper demonstrate that the structural gene present in Dm 225 DNA is a representative of a multiple gene family dispersed throughout the whole genome of D.melanogaster.Images

Animals↗
PubMed1978

Chromosome of the mature virion of simian virus 40 contains H1 histone.

In addition to free SV40 minichromosomes in the compact form, complete virions were obtained from the nuclear extract of productively infected cells. Capsid proteins VP1, VP2, and VP3, as well as histones, were observed on electrophoregrams of proteins prepared from virions. In contrast to the widely accepted view, histone H1 was found in virions in stoichiometric amounts with respect to other histones. The same is true for virions isolated by a conventional method. Free minichromosomes present in infected cells contain all histones and practically no viral proteins.

Chromatin↗
PubMed1977

Isolation of eukaryotic DNA fragments containing structural genes and the adjacent sequences.

In Drosophila melanogaster structural genes are located close to moderately reiterated sequences. One of the clones obtained contains the DNA related to intercalary heterochromatin of D. melanogaster. These are individual differences in the distribution of genetic material in polytenic chromosomes of different stocks of D. melanogaster. The techniques that allow isolation of DNA fragments containing structural genes at the beginning, in the middle, or the end of the coding strand have been elaborated.

Animals↗
PubMed1977

Compact form of SV40 viral minichromosome is resistant to nuclease: possible implications for chromatin structure.

We report two new findings bearing on the "supranucleo-somal" level of the structure of the Simian Virus 40 minichromosome. I) Isolated SV40 minichromosome which contains all five histones including HI/I/ exists in solution under approximately physiological ionic conditions as a compact roughly spherical particle approximately 300 A in diameter which is capable of fitting within the virus capsid. In spite of such a compact conformation of the minichromosome individual nucleosomes can be readily visualized within the particle. Compact state of SV40 minichromosome depends on both the presence of histone HI and maintenance of approximately physiological ionic strength of solution (micron approximately 0.15). Removal of HI results in a conversion of the compact minichromosomes into an extended (circular beaded) structure. 2) The compact form of the SV40 minichromosome in contract to its circular beaded form is virtually completely resistant to staphylococcal nuclease, strongly suggesting that in particular nuclease-sensitive parts of the internucleosomal DNA regions are not exposed on the outside of the compact SV40 minichromosome. On the other hand, DNase I which is known to attack both inter-and intranucleosomal DNA in the chronatin /2,3/ readily digests the compact form of the SV40 minichromosome. Possible models of the compact minichromosome and implications for higher order structures of the cellular chromatin are discussed.

Centrifugation, Density Gradient↗
PubMed1977

Histone-like proteins in the purified Escherichia coli deoxyribonucleoprotein.

Analysis of E.coli chromosomes isolated under conditions similar to those used for isolation of eukaryotic chromatin has shown that: 1) The proteins of highly purified E.coli deoxyribonucleoprotein are mainly in addition to RNA polymerase two specific histone-like proteins of apparent molecular weight of 17,000 and 9,000 (proteins 1 and 2, respectively). 2) Proteins 1 and 2 occur in approximately equal molar amounts in the isolated E.coli chromosome, and their relative content corresponds to one molecule of protein 1 plus one molecule of protein 2 per 150-200 base pairs of DNA. 3) There are no long stretches of naked DNA in the purified E.coli deoxyribonucleoprotein suggesting a fairly uniform distribution of the proteins 1 and 2 along DNA. 4) The protein 2 is apparently identical to the DNA-binding protein HU which was isolated previously /1/ from extracts of E.coli cells. 5) Digestion of the isolated E.coli chromosomes with staphylococcal nuclease proceeds through discrete deoxyribonucleoprotein intermediates (in particular, at approximately 120 base pairs) which contain both proteins 1 and 2. However, since no repeating multimer structure was observed so far in nuclease digests of the E.coli chromosome, it seems premature to draw definite conclusions about possible similarities between the nucleosomal organization of the eukaryotic chromatin and the E.coli chromatin structure.Images

Chromosomes, Bacterial↗
PubMed1976

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↗
PubMed1976

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↗
PubMed1976

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↗