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Biomedical subjects

O P Samarina

Publications and source records attributed to O P Samarina.

At least 19 recordsLinked to original sources

Insertions of hybrid P elements in the yellow gene of Drosophila cause a large variety of mutant phenotypes.

A series of yellow mutations associated with a great variety of tissue-specific phenotypes were obtained from several highly unstable Drosophila melanogaster strains carrying the gypsy-induced y2 allele. These mutations are caused by insertion of additional DNA sequences of variable size 69 bp upstream of the yellow transcription start site. These sequences are flanked by identical copies of a deleted 1.2-kb P element arranged in the same or inverted orientation. The central part of the inserted element consists of genomic sequences originating from different regions of the X chromosome. The mutant phenotype caused by these chimeric elements depends on the nature of the sequences present either in the P element or in the central part of the insertion, suggesting that these sequences are able to affect expression of the yellow gene. In addition, sequences present in the central region of the insertions strongly modify the effects of the gypsy-bound suppressor of Hairy-wing [su(Hw)] and modifier of mdg4 [mod(mdg4)] proteins on yellow transcription. Analyses of these mutations give new insights into the mechanisms by which su (Hw) and mod(mdg4) affect enhancer function.

Alleles↗

P element-flanked inserts at the yellow locus of Drosophila melanogaster strains.

Large inserts have been found to appear within the yellow (y) locus of several unstable mutant lines derived from a parental strain (y2 sc+ w(aG)) of Drosophila melanogaster. The inserts of the individual mutant lines correspond roughly to a series of multimers (4.1, 7.1, 14.3 and 28.9 kbp, respectively) and are always located at the same position, somewhat upstream of the y gene transcription start site. Each of them begins and ends with a 1-kbp sequence which shares restriction sites with P elements. One of these inserts (14.3 kbp) has been partially sequenced (1270 bp from the 5' end). The first 1185 bp match exactly the sequence of an internally deleted P element. The remaining part of the sequence, on the other hand, is unrelated to P elements. It is assumed that large DNA sequences may be captured by the flanking P-element fragments and inserted into new chromosomal sites.

Animals↗

[Tissue-specific blocking of the EcoRI site adjacent to the pseudogene for mouse oncoprotein p53].

EcoRI fragments of DNA isolated from the different mouse organs were hybridized to radioactivity labelled probe specific for the gene of oncoprotein p53. The analysis of the blot-hybridization points to the existence of the specific blockage of an EcoRI site flanking a 3.3 kb fragment of DNA including the pseudogene p53, isolated from the skin tissue. The existence of a polymorphous EcoRI site localized distally to the pseudogene p53 has been demonstrated in the DNA of mice of different lines.

Animals↗

A variation in the structure of the protein-coding region of the human p53 gene.

An extensive analysis of genomic DNA preparations from a number of normal and malignant tissues revealed BglII site polymorphism of the human p53 gene. Approximately 10% of p53 gene alleles were found to contain an additional BglII site localized in a region of intron I. This allelic form of p53 gene was also responsible for p53 protein having altered electrophoretic mobility. Molecular cloning and sequencing of both the alleles of p53 gene revealed a base-pair change in codon 72 causing arginine----proline substitution in the allele with the additional BglII site. Both variants of the p53 gene may occur in homozygous state and are therefore functional.

Amino Acid Sequence↗

[Structural organization of the human p53 gene. I. Molecular cloning of the human p53 gene].

Human p53 gene was cloned from the normal human placenta DNA and DNA from the strain of human kidney carcinoma transplanted into nude mice. Representative gene library from tumor strain of human kidney carcinoma and library of 15 kb EcoRI fragments of DNA from normal human placenta were constructed. Maniatis gene library was also used. Five clones were isolated from kidney carcinoma library; they covered 27 kb and included full-length p53 gene of 19.5 kb and flanking sequences. From normal placenta libraries three overlapped clones were obtained. Restriction map of cloned sequences was constructed and polarity of the p53 gene determined. The first intron of the gene is large (10.4 kb); polymorphic BglII site was observed in this intron, which allows to discriminate between allelic genes. One of these (BglII-) is ten times more abundant that the other (BglII+). Both allelic genes are able to synthesize the 2.8 kb p53 gene.

Animals↗

The sequences homologous to major interspersed repeats B1 and B2 of mouse genome are present in mRNA and small cytoplasmic poly(A) + RNA.

Heavy nuclear RNA contains high amounts of transcripts from repetitive sequences B1 and B2. Cytoplasmic poly(A)+RNA and, particularly, polysomal poly(A)+RNA (mRNA) also include these sequences but in smaller amounts. The abundant 2 kb mRNA of mouse liver are found to have a B2 sequence. These sequences are also found in a new class of low-molecular-weight heterogeneous (200-400 nucleotide long) poly(A)+RNAs. These RNAs are located mostly in cytoplasm rather than in polysomes. The amount of small B2+ RNAs is noticeable larger than that of small B1+ RNAs. Tumour cells seem to contain more small B2 RNA than normal cells. The hybridization tests show that extended homology exists between the B1 sequence and 4.5S small nuclear RNA, which is predicted from comparison of their base sequences. Also, we have found homology between B1 and small cytoplasmic 7S RNA. Hybridization of B2 to sn or sc RNAs has not been observed, although the sequencing reveals partial homology between B2 and 4.5S sn RNAI /1/.

Animals↗

Structural investigation of nuclear RNP particles containing pre-mRNA by different fluorescence techniques.

Ethidium bromide (EB) adsorption isotherms on 30S nuclear RNP particles isolated from liver nuclei has revealed 6% of double-stranded regions in pre-mRNA (dsRNA). It has been established by measurements of the EB fluorescence polarization that the bulk of dsRNA regions in RNP is rigidly attached to RNP. They are longer than 45 degree A. The increase of NaCl concentration from 0.1 up to 0.4 M causes a significant loosening of dsRNA-protein bonds. As a result the dsRNA segments become more flexible. Measurements of energy transfer from fluorescamine (covalently bound to the protein) to EB (adsorbed on dsRNA) have yielded information about dsRNA location. The fact that absorbtion of exciting light by fluorescamine causes pronounced increase of EB fluorescence is consistent with the idea that helical regions of RNA are located outside the RNP particles.

Animals↗

[Structure of nuclear pre-mRNA. XIII. Hybridization properties of triphosphorylated 5'-end fragments].

Triphosphorylated 5'-end fragments about 100 nucleotides long were prepared from purified nuclear pre-mRNA using a modified hydroxyapatite method. These fragments as well as fragments of total pre-mRNA were polyadenylated by ATP:RNA adenyl-transferase and used as templates for the synthesis of [32P]cDNA by reverse transcriptase in the presence of an oligo(dT)-primer, cDNA transcribed from total fragments of pre-mRNA and from 5'-end fragments (5'-cDNA) were hybridized with excess of nuclear pre-mRNA. The extent of hybridization was 65-70 and 80-85% in different experiments. 18% of total cDNA and 35% of 5'-cDNA hybridized with mRNA from polysomes. A high homology between mRNA and triphosphorylated 5'-ends of pre-mRNA may be explained in the terms of splicing. The sequences adjacent to the triphosphorylated 5'-ends of pre-mRNA represent a specific class with complexity about 2.10(5) nucleotides. Less than 30% of 5'-cDNA hybridized with intermediately repetitive DNA, while the main portion hybridized with unique DNA sequences. About 15% of 5'-cDNA contain oligo (dA) sequences, originated from oligo (U) in the pre-mRNA.

Adenosine Triphosphate↗

Cross-linked informofers.

The proteins of 30S RNP particles containing pre-mRNA (hnRNA) were cross-linked with bifunctional reagents (dimethyl-suberimidate and dimethyl-3,3'-dithiobispropionimidate). Further treatment with 1 or 2 M NaCl dissociates all RNA from protein. However, a significant part of protein particles--informofers being cross-linked survived high salt treatment. Their sedimentation coefficients were close to those of original particles. No RNA could be detected in the informofers even after labeling the cells with a precursor for a long period of time. Sodium dodecylsulfate or urea dissociated cross-linked informofers into oligomeric polypeptides. They could be dissociated by beta-mercaptoethanol treatment if a reversible cross-linked reagent had been used. The resulting polypeptides were represented by informatin. RNP particles (30S RNP or poly-particles) were reconstituted upon mixing of cross-linked informofers with pre-mRNA and removal of 2 M NaCl.

Animals↗

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↗

The properties of oligonucleotide fragments containing the triphosphorylated 5'-termini of nuclear pre-mRNA from Ehrlich ascites carcinoma cells.

Triphosphorylated 5'-end fragments 50-150 nucleotides in length were isolated from nuclear pre-mRNA with the aid of a hydroxyapatite chromatography. They are enriched in U and G (28 and 26%, respectively). About 15% of the fragments isolated from poly(U)+RNA contain poly(U) tracks. Neither poly(A)- nor double-stranded sequences were found. Hybridization experiments in conditions of vast DNA excess demonstrated that the 5'-end fragments contain a low amount of highly repetitive sequences but enriched in sequences hybridizing at C0t 1/2 approximately 100.

Animals↗

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↗

Autodegradation of pre-mRNA containing nuclear ribo-nucleoprotein particles. The effect of autodegradation on the double-stranded RNA sequences and on the protein composition of particles.

The quantitative changes of double-stranded RNA components of nuclear ribonucleo-protein particles containing pre-mRNA was investigated in the course of incubation of particles at 37 degrees C. The incubation of purified nuclear particles revealed the fragmentation of long double-stranded RNA sequences into shorter stretches. The presence of nuclear sap in the incubation mixture resulted in degradation of the double-stranded RNAs into acid soluble products. Autodegradation and/or ribonuclease treatment of nuclear RNP particles is accompanied by quantitative changes in the minor protein constituents of informofer.

Animals↗

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↗

The poly(A)-containing ribonucleoproteins in the nucleus and the cytoplasm of ehrlich ascites carcinoma cells.

The sedimentation characteristics and protein composition of the polyadenylic acid-containing ribonucleoprotein (RNP) particles separated from the polysomes of Ehrlich ascites carcinoma cells by means of ribonuclease treatment were studied. The results were compared with the corresponding characteristics of the poly(A)-containing particles of the cell nucleus. The sedimentation coefficient of the poly(A)-containing RNP from poly-ribosomes was found to be 11 S, while that of the nuclear particles was 14 S. In Cs2SO4 equilibrium density gradient values of p=1.25 and 1.34 gcm-3 were obtained for the nuclear particles and the poly(A)-RNP separated from polyribosomes, respectively. The molecular weights of the proteins making up the polysomal poly(A)-particles were found to be over 40 000 daltons, i.e. 54 000, 48 000, 68 000 and 89 000 daltons for the four polypeptides present in considerable amounts. As far as electrophoretic mobility is concerned these proteins were indistinguishable from the proteins of the nuclear poly(A)-containing RNP. However, the ratio of the components in the two types of particle proved to be quite different.

Animals↗

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↗

Protein composition of nuclear 14 S ribonucleoprotein particles containing poly (A).

Nuclear 14 S RNP particles containing poly (A) from Ehrlich ascites carcinoma cells and rat liver were purified by re-sedimentation in sucrose gradients, by Cs2SO4 density gradient centrifugation and by affinity chromatography on a poly (dT)-Sepharose column. Proteins of these RNP particles were electrophoresed in urea and SDS-polyacrylamide gels. RNP particles of ascites carcinoma cells contain two main bands having molecular weights of 51 000 and 69 000 daltons, respectively, and two or three minor components.

Adenine Nucleotides↗