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D K Biswas

Publications and source records attributed to D K Biswas.

At least 55 records · Page 3Linked to original sources
PubMed1983

Extent of BrdUrd-induced prolactin gene amplification in GH cells.

The thymidine analog, 5-bromodeoxyuridine (BrdUrd), induces prolactin (Prl) synthesis and Prl gene amplification in a subclone of GH cells (rat pituitary tumor cells in culture). Withdrawal of drug treatment reverses both processes. Our previous results show that Prl gene amplification is associated with an event involving extrachromosomal DNA. The results presented in this report show that a 20-kb length of DNA, including all the coding sequences of the Prl gene, is amplified following BrdUrd treatment of this strain of GH cells. Amplification of DNA sequences extends about 3 kb upstream from the 5' end and about 7 kb downstream from the 3' end of the Prl gene. The DNA sequences immediately adjacent to these 5'-and 3'-flanking regions of the Prl gene are not affected by BrdUrd treatment. These results suggest that BrdUrd-induced amplification of Prl gene coding and neighboring sequences is restricted to within 3 kb of the 5' end and within 7 kb of the 3' end of the structural gene Analysis of low-molecular-weight extrachromosomal DNA isolated by Hirt's procedure from drug-treated cells reveals a similar pattern of amplification of the Prl gene and its flanking sequences.

Animals↗
PubMed1982

Increased level of prolactin gene sequences in bromodeoxyuridine treated GH cells.

The 5-bromodeoxyuridine-resistant (BrdUrdr) derivative (F1BGH12C1) of prolactin nonproducing (PRL-) rat pituitary tumor cell-subclone GH12C1, synthesize prolactin (PRL) in the presence of the drug. Analysis of nuclear RNA isolated from BrdUrd treated F1BHG12C1 cells demonstrated several high molecular weight RNA PRL sequences, similar to those observed in the nuclear RNA fraction of PRL producing (PRL+) GH3 cells. No such RNAPRL sequences could be detected in nuclear RNA fraction of untreated F1 BGH12C1 cells. PRL sequences in the genome of GH3 (PRL+), GH12C1 (PRL-) and F1BGH12C1 (PRL-, BrdUrdr) GH cells could be identified by blot analysis in 4.8-5.2kb fragment of restriction endonuclease, Hind III digested DNA. Both PRL+ and PRL- cells seem to have approximately the same level of PRL gene sequences in total cell DNA. However Hind III digested DNA of BrdUrd treated F1BGH12C cells revealed the presence of significantly higher levels of PRL gene sequences, in comparison, to that observed in total DNA of untreated cells. The increased level of PRL gene sequences was dependent on the period of drug treatment and a parallel increase in the cytoplasmic RNAPRL sequences was also observed.

Animals↗
PubMed1982

Mechanism of induction of prolactin synthesis in GH cells.

Prolactin-specific RNA (RNA(PRL)) in total nuclear RNA and in cytoplasmic poly(A)(+)RNA isolated from GH (rat pituitary) cells was selectively hybridized to immobilized cloned cDNA(PRL). Agarose gel electrophoresis of the nuclear RNA(PRL) sequences eluted from the nitrocellulose filters revealed several RNA species of approximately 25-30, 18-19, and 12-13 S. Only the 12-13 S RNA species could be detected in the cytoplasmic poly(A)(+)RNA fraction. Comparative analysis of total nuclear RNA of control and thyrotropin-releasing hormone (thyroliberin)-treated cells by the reverse Southern blot technique demonstrated increased levels of all the nuclear RNA(PRL) species in hormone-treated cells. Nuclear and cytoplasmic RNA(PRL) sequences in control and treated cells were quantitated by molecular hybridization to cloned cDNA(PRL). The 2- to 3-fold stimulation of PRL production by thyrotropin-releasing hormone-treated GH(4)C(1) cells could be correlated to the corresponding increase of nuclear RNA(PRL) sequences. The hybrid strain, which produces 1/5th the amount of PRL that the parent GH(4)C(1) does, had 1/5th the amounts of nuclear RNA(PRL) sequences. Thyrotropin-releasing hormone affected neither prolactin production nor nuclear RNA(PRL) level in 928-9b cells. RNA(PRL) sequences could not be detected either in nuclei or in cytoplasm of prolactin nonproducing F(1)BGH(1)2C(1) cells. However, prolactin production could be induced and RNA(PRL) sequences could be detected in the total nuclear RNA and in cytoplasmic poly(A)(+)RNA fraction after treatment of this GH cell substrain with 5-bromodeoxyuridine. These results demonstrate that differential basal prolactin production and its modulation by thyrotropin-releasing hormone and by 5-bromodeoxyuridine can be correlated to the altered levels of nuclear RNA(PRL) sequences in the three GH cell strains.

Animals↗
PubMed1980

Regulated expression of the prolactin gene in rat pituitary tumor cells.

Prolactin (PRL) gene expression in three strains of GH cells (rat pituitary tumor cells) has been quantitated by measurement of: (a) intracellular and extracellular PRL, (b) cytoplasmic translatable PRL-specific mRNA (mRNAPRL), and (c) molecular hybridization of cytoplasmic poly(A) RNA to cDNAPRL (DNA complementary to mRNAPRL). Three GH cell lines utilized in this investigation were a PRL-producing (PRL+) strain, GH4C1, a PRL nonproducing 5-bromo-deoxyuridine resistnat (PRL- BrdUrdr) strain, F1BGH12C1, and a new strain, 928-9b, derived by fusion of PRL+ cells with a nuclear monolayer of the PRL-, BrdUrdr GH cell strain. PRL production is a characteristic of 928-9b cells, but the level of PRL production (2-4 micrograms/mg protein/24 h) is much lower than that of the PRL+ strain, GH4C1 (15-25 micrograms/mg protein/24 h). Levels of cytoplasmic translatable mRNAPRL and cytoplasmic PRL-RNA sequences quantitated with a cDNAPRL probe were also much lower in 928-9b as compared to the PRL+ parent. PRL-RNA sequences could not be detected in the PRL- strain. Thyrotopin-releasing hormone (TRH) stimulates PRL synthesis about threefold and inhibit a growth hormone (GH) synthesis 72% in the PRL+ strain. TRH has no effect on the synthesis of either PRL or GH in the 928-9b strain, although TRH receptors could be detected in these cells. Stimulation of PRL synthesis in the PRL+ strain by TRH could be correlated with increases in levels of cytoplasmic translatable mRNAPRL and increases in cytoplasmic PRL-RNA sequences. These results demonstrate that the graded expression of the PRL gene at the basal level, and in response to TRH, is caused by the regulated production of specific mRNA, i.e., mRNAPRL in these three GH cell strains.

Animals↗
PubMed1979

On the mechanism of 5-bromodeoxyuridine induction of prolactin synthesis in rat pituitary tumor cells.

GH12C1, a clonal strain of rat pituitary tumor cells in culture (GH cells), does not produce detectable amounts of prolactin. 5-Bromodeoxyuridine (BrdUrd), the thymidine analogue, at sublethal concentrations (3-5 microgram/ml) induces prolactin synthesis in these cells. BrdUrd also induces prolactin synthesis in F1BGH12C1 cells, a BrdUrd resistant (BrdUrdr) substrain isolated from GH12C1 cells. The F1BGH12C1 strain is not drug dependent, but its resistance to BrdUrd is a stable phenotype. The significant features of the induction of prolactin synthesis in the BrdUrdr strain are the increased net synthesis of prolactin and the shortening of the lag period of prolactin induction. As BrdUrd concentration in the growth medium is increased, the rise in prolactin synthesis parallels the increased incorporation of BrdUrd into DNA. Prolactin synthesis is first detected when BrdUrd replaces 20-25% of the thymidine in DNA. BrdUrd can replace up to 75-80% of the thymidine within 2 d of treatment. Partial starvation of these cells under specified growth conditions does not affect the general growth pattern of the cells, general protein synthesis, and thymidine uptake, but does affect DNA synthesis. When cells are cultured under conditions in which DNA synthesis is preferentially inhibited, BrdUrd does not induce prolactin synthesis, suggestive of a DNA-mediated mechanism of action for the drug.

Animals↗
PubMed1976

Extended RNA synthesis in isolated nuclei from rat pituitary tumor cells.

Nuclei of GH3 cells, isolated by detergent lysis, synthesized RNA for an extended period at 29 degrees C in the presence of rat liver ribonuclease inhibitor (RI). Extended RNA synthesis was dependent upon the presence of RI. Sucrose gradient sedimentation analysis of the cell-free reaction products showed that RNAs ranging from 4 S to greater than 28 S were synthesized. Further characterization of the RNA products was made by examining the sensitivity of synthesis to alpha-amanitin and actinomycin D as well as by oligo(dT)-cellulose binding properties. Evidence was obtained that RNA polymerases I, II, and III were functioning in isolated GH3 nuclei. Newly synthesized RNAs were found in both the nuclear pellet and postnuclear supernatant fractions. RNA polymerase I products remained associated with the nuclear pellet throughout a 60-min incubation period whereas RNAs synthesized by RNA polymerase III emerged rapidly into the supernatant fraction. RNA polymerase II products were distributed in both fractions and were found to contain poly(A). De novo poly(A) synthesis was demonstrated and found to be inhibited by cordvcepin triphosphate (3'-dATP). Supernatant RNAs synthesized by polymerase II contained a poly(A) segment of about 150 adenine residues; these transcripts sedimented heterogeneously with an apparent size distribution (under denaturing conditions) which was smaller than that of nuclear RNA polymerase II products and which resembled that of cellular mRNA. Qualitative differences in the nuclear and supernatant RNAs, the kinetics of appearance of the latter, and the differential effect of 3'-dATP on the extranuclear appearance of supernatant RNAs suggest that a process resembling nuclear-cytoplasmic RNA transport occurred in this cell-free nuclear system.

Adenosine Triphosphate↗
PubMed1974

The effects of streptomycin or dihydrostreptomycin binding to 16S RNA or to 30S ribosomal subunits.

Evidence is presented suggesting that streptomycin binds to 16S RNA or to 30S ribosomal subunits at the same topographical site located on the RNA chain. The equally bactericidal dihydrostreptomycin binds to the same site as streptomycin but with lower affinity. The effect of drug binding to 16S RNA (measured by reconstitution inhibition) is readily reversible, while that of drug binding to 30S subunits (measured by misreading) persists after removal of the drug. Binding of the drug is not a necessary and sufficient reason for killing.

Carbon Radioisotopes↗
PubMed1972

The attachment site of streptomycin to the 30S ribosomal subunit.

The 16S RNA dissociated from 30S ribosomal subunits of Escherichia coli strains either sensitive or resistant to streptomycin contains the attachment sites for two streptomycin molecules, as does the undissociated particle from a streptomycin-sensitive strain. Since no streptomycin binds to undissociated 30S subunits from a streptomycin-resistant strain, it is suggested that protein P10, specified by the strA locus-known to be responsible for drug sensitivity-controls the availability to streptomycin of the attachment sites. These sites remain exposed in the strA(+) wild-type, and become masked in strA streptomycin-resistant mutants. The 16S RNA molecule binds streptomycin specifically; it binds two drug molecules in its native state, binds many more after its secondary structure is unfolded by melting out, and again binds two molecules after reannealing. The binding is stable to exhaustive dialysis, but it is reversed by exposure of the streptomycin-RNA complex to high-salt concentration. The complex can not be used to reconstitute functional ribosomes, but the 16S RNA reacquires this property after streptomycin elimination. The biological significance of this stable streptomycin binding is questioned, since in strA mutants exhibiting phenotypic masking, exposure to streptomycin induces a modified 30S behavior that persists even after streptomycin has been dialyzed away, and is reversed only by exposure of the modified RNA to high-salt concentration.

Binding Sites↗