Specific polyadenylation and purification of total messenger RNA from Escherichia coli

Obtaining pure mRNA preparations from prokaryotes has been difficult, if not impossible, for want of a poly(A) tail on these messages, We have used poly(A) polymerase from yeast to effect specific polyadenylation of Escherichia coli polysomal mRNA in the presence of magnesium and manganese, The polyadenylated total mRNA, which could be subsequently purified by binding to and elution from oligo(dT) beads, had a size range of 0.4-4.0 kb. We have used hybridization to a specific plasmid-encoded gene to further confirm that the polyadenylated species represented mRNA, Withdrawal of Mg2+ from the polyadenylation reaction rRNA despite the presence of Mn2+, indicating the vital role of Mg2+ in maintaining the native structure of polysomes, Complete dissociation of polysomes into ribosomal subunits resulted in quantitative polyadenylation of both 16S and 23S rRNA species, Chromosomal lacZ gene-derived messages were quantitatively recovered in the oligo(dT)-bound fraction, as demonstrated by RT-PCR analysis, Potential advantages that accrue from the availability of pure total mRNA from prokaryotes is discussed.

Dexamethasone negatively regulates phenobarbitone-activated transcription but synergistically enhances cytoplasmic levels of cytochrome P-450b/e messenger RNA

Dexamethasone has a potentiating effect on phenobarbitone mediated induction of cytochrome P-450b + e mRNAs in adult rat liver. However, the glucocorticoid inhibits phenobarbitone-activated transcription of cytochrome P-450b + e mRNAs by 60-70%. This inhibitory effect is evident in run-off transcription of the endogenous genes as well as in the transcription of an added cloned gene fragment. Dexamethasone inhibits the phenobarbitone-mediated increase in the binding of a transcription factor(s) to the upstream region of the gene as evidenced by gel retardation and Southwestern blot analysis. The glucocorticoid does not stabilize the phenobarbitone-induced polyribosomal cytochrome P-450b + e mRNAs but appears to stabilize the nuclear transcripts. It is proposed that a negative element may mediate the action of dexamethasone at the level of nuclear transcription and stabilization of the nuclear transcript may account for the potentiating effect of the glucocorticoid on phenobarbitone-mediated increase in cytochrome P-450b + e mRNAs in the cytoplasm of the adult rat liver. However, the cytochrome P-450b protein levels are slightly lower in phenobarbitone + dexamethasone treatment than in phenobarbitone-treated liver microsomes.

Perturbation of nucleo-cytoplasmic transport affects size of nucleus and nucleolus in human cells

Size regulation of human cell nucleus and nucleolus are poorly understood subjects. 3D reconstruction of live image shows that the karyoplasmic ratio (KR) increases by 30-80% in transformed cell lines compared to their immortalized counterpart. The attenuation of nucleo-cytoplasmic transport causes the KR value to increase by 30-50% in immortalized cell lines. Nucleolus volumes are significantly increased in transformed cell lines and the attenuation of nucleo-cytoplasmic transport causes a significant increase in the nucleolus volume of immortalized cell lines. A cytosol and nuclear fraction swapping experiment emphasizes the potential role of unknown cytosolic factors in nuclear and nucleolar size regulation.

17 beta-Estradiol and progesterone improve in-vitro cytoplasmic maturation of oocytes from unstimulated prepubertal and adult rhesus monkeys

BACKGROUND: Effects of 17beta-estradiol and progesterone on rhesus monkey oocyte maturation in vitro were evaluated by embryo development subsequent to IVF. METHODS AND RESULTS: In experiment 1, immature cumulus-oocyte complexes collected from unstimulated adult females during the non-breeding season were matured in modified medium CMRL-1066 containing various combinations of gonadotrophins (FSH + LH), estradiol and/or progesterone. Formation of morulae and blastocysts was greatest in oocytes matured in medium containing estradiol and/or progesterone, with or without gonadotrophins (morula 38-46%, blastocyst 14-20%) than in control oocytes matured without estradiol or progesterone (morula 14%, blastocyst 0%). In experiment 2, cumulus-oocyte complexes from unstimulated prepubertal female monkeys were matured in medium with gonadotrophins, estradiol or progesterone. The best development to the morula stage was obtained with oocytes matured with gonadotrophins and estradiol or gonadotrophins and progesterone (43 and 25 morulae, respectively), while control oocytes matured with gonadotrophins but without steroid hormones gave the poorest morula developmental response (12%). However, there was no difference in blastocyst development across all groups (0-3%). CONCLUSIONS: These results demonstrate that during rhesus monkey oocyte maturation in vitro: (i) estradiol or progesterone can improve oocyte developmental competence; (ii) immature oocytes from prepubertal versus adult females have differential responses to challenge with estradiol or progesterone.

Secondary structure of yeast genomic downstream region and polyadenylation signals

Polyadenylation of 3 ' -forming in eukaryote concerns three elements located in precursor mRNA downstream region: efficiency element (EE), position element (PE) and the actual site for cleavage and polyadenylation. Several base sequences of EE and PE have

Rana grylio virus thymidine kinase gene: an early gene of iridovirus encoding for a cytoplasmic protein

The presence of thymidine kinase (TK) is a feature of many large DNA viruses. Here, a TK gene homologue was cloned and characterized from Rana grylio virus (RGV), a member of family Iridoviridae. RGV TK encodes a protein of 195 aa with a predicted molecular mass of 22.1 kDa. Homologues of the protein were present in all the currently sequenced iridoviruses, and phylogenetic analysis showed that it was much close to cellular TK type 2 (TK2), deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Subsequently, Western blotting revealed TK expression increased with time from 6 h post-infection in RGV-infected cells. Using drug inhibition analysis by protein synthesis inhibitor (cycloheximide) and DNA replication inhibitor (cytosine arabinofuranoside), RGV TK was classified as the early expression gene during in vitro infection. Subcellular localization by TK-GFP fusion protein expression and immunofluorescence staining showed RGV TK was an exclusively cytoplasmic protein in fish cells. Collectively, current data indicate that RGV TK was an early gene of iridovirus which encoded a cytoplasmic protein in fish cells.

Construction of cytoplasmic molecular markers distinguishing Danio rerio from Gobiocypris rarus at high identity domains based on MP-PCR strategy and Sybr Green I detection

To distinguish the cytoplasm of Danio rerio from that of Gobiocypris rarus, we cloned G. rarus COXI and constructed cytoplasmic molecular markers at the high identity domains of COXI by mutated primer PCR (MP-PCR for short). Then Sybr Green I was used to detect the single amplicon. As a result, we succeeded in getting the cytoplasmic molecular markers, G.M COXI and Z.M COXI, by MP-PCR strategy. They were used to detect the sperm-derived mtDNA in the sexual hybrid embryos (D. rerio female x G. rarus male) before the sphere stage. In the present study, all results demonstrate that MP-PCR approach and Sybr Green I detection are feasible to construct the molecular markers to identify genes that shared high identity.

Cytoplasmic impact on cross-genus cloned fish derived from transgenic common carp (Cyprinus carpio) nuclei and goldfish (Carassius auratus) enucleated eggs

In previous studies of nuclear transplantation, most cloned animals were obtained by intraspecies nuclear transfer and are phenotypically identical to their nuclear donors; furthermore, there was no further report on successful fish cloning since the report of cloned zebrafish. Here we report the production of seven cross-genus cloned fish by transferring nuclei from transgenic common carp into enucleated eggs of goldfish. Nuclear genomes of the cloned fish were exclusively derived from the nuclear donor species, common carp, whereas the mitochondrial DNA from the donor carp gradually disappeared during the development of nuclear transfer (NT) embryos. The somite development process and somite number of nuclear transplants were consistent with the recipient species, goldfish, rather than the nuclear donor species, common carp. This resulted in a long-lasting effect on the vertebral numbers of the cloned fish, which belonged to the range of goldfish. These demonstrate that fish egg cytoplasm not only can support the development driven by transplanted nuclei from a distantly related species at the genus scale but also can modulate development of the nuclear transplants.

The interaction of aldolase with the cytoplasmic domain of human erythrocyte band 3 inhibited by lanthanum ions

The effect of lanthanum ions on the activity of the cytoplasmic domain of human erythrocyte band 3 (CDB3), which was measured according to the inhibition to aldolase, was studied. In the presence of low concentration of lanthanum ions, the function of CDB3 to inhibit aldolase activity decreased significantly. It indicated that lanthanum ions in the erythrocyte would change the conformation of CDB3 and influence the control on aldolase activity.