Comparison of in vivo and in vitro RNA synthesis on polytene chromosomes of Drosophila

Chromosoma ◽  
1976 ◽  
Vol 54 (4) ◽  
pp. 349-362 ◽  
Author(s):  
B. A. Leibovitch ◽  
E. S. Belyaeva ◽  
I. F. Zhimulev ◽  
R. B. Khesin
Keyword(s):  
Rna Synthesis ◽  
Polytene Chromosomes

Studies of two temperature-sensitive mutants of Mengo virus

1979 ◽  
Vol 57 (6) ◽  
pp. 902-913 ◽  
Author(s):  
Patrick W. K. Lee ◽  
John S. Colter
Keyword(s):  
Rna Synthesis ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Supporting Evidence ◽  
Structural Polypeptide ◽  
Infected Cells ◽  
Mengo Virus ◽  
In Vitro Stability

Studies of the synthesis of viral ribonucleates and polypeptides in cells infected with two RNA−ts mutants of Mengo virus (ts 135 and ts 520) have shown that when ts 135 infected cells are shifted from the permissive (33 °C) to the nonpermissive (39 °C) temperature: (i) the synthesis of all three species of viral RNA (single stranded, replicative form, and replicative intermediate) is inhibited to about the same extent, and (ii) the posttranslational cleavage of structural polypeptide precursors A and B is partially blocked. Investigations of the in vivo and in vitro stability of the viral RNA replicase suggest that the RNA− phentotype reflects a temperature-sensitive defect in the enzyme. The second defect does not appear to result from the inhibition of viral RNA synthesis at 39 °C, since normal cleavage of polypeptides A and B occurs in wt Mengo-infected cells in which viral RNA synthesis is blocked by cordycepin, and at the nonpermissive temperature in ts 520 infected cells. Considered in toto, the evidence suggests that ts 135 is a double mutant.Subviral (53 S) particles have been shown to accumulate in ts 520 (but not ts 135) infected cells when cultures are shifted from 33 to 39 °C. This observation provides supporting evidence for the proposal that this recently discovered particle is an intermediate in the assembly pathway of Mengo virions.

scholarly journals The closely related Drosophila sry beta and sry delta zinc finger proteins show differential embryonic expression and distinct patterns of binding sites on polytene chromosomes

Development ◽  
1990 ◽  
Vol 110 (1) ◽  
pp. 141-149 ◽  
Author(s):  
F. Payre ◽  
S. Noselli ◽  
V. Lefrere ◽  
A. Vincent
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Polytene Chromosomes ◽  
Duplication Event ◽  
Amino Acid Sequences ◽  
Evolutionary Divergence ◽  
Coding Region ◽  
Protein Coding

Serendipity (sry) beta (beta) and delta (delta) are two finger protein genes resulting from a duplication event. Comparison of their respective protein products shows interspersed blocks of conserved and divergent amino-acid sequences. The most extensively conserved region corresponds to the predicted DNA-binding domain which includes 6 contiguous fingers; no significant sequence conservation is found upstream and downstream of the protein-coding region. We have analysed the evolutionary divergence of the sry beta and delta proteins on two separate levels, their embryonic pattern of expression and their DNA-binding properties in vitro and in vivo. By using specific antibodies and transformant lines containing beta-galactosidase fusion genes, we show that the sry beta and sry delta proteins are maternally inherited and present in embryonic nuclei at the onset of zygotic transcription, suggesting that they are transcription factors involved in this process. Zygotic synthesis of the sry beta protein starts during nuclear division cycles 12–13, prior to cellularisation of the blastoderm, while the zygotic sry delta protein is not detectable before germ band extension (stage 10 embryos). Contrary to sry delta, the zygotic sry beta protein constitutes only a minor fraction of the total embryonic protein. The sry beta and delta proteins made in E. coli bind to DNA, with partly overlapping specificities. Their in vivo patterns of binding to DNA, visualised by immunostaining polytene chromosomes, differ both in the number and position of their binding sites. Thus changes in expression pattern and DNA-binding specificity have contributed to the evolution of the sry beta and delta genes.

scholarly journals Interaction between the DrosophilaCAF-1 and ASF1 Chromatin Assembly Factors

2001 ◽  
Vol 21 (19) ◽  
pp. 6574-6584 ◽  
Author(s):  
Jessica K. Tyler ◽  
Kimberly A. Collins ◽  
Jayashree Prasad-Sinha ◽  
Elizabeth Amiott ◽  
Michael Bulger ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Polytene Chromosomes ◽  
Normal Growth ◽  
Chromatin Assembly ◽  
Truncated Form ◽  
Assembly Factor ◽  
Development And Differentiation ◽  
Growth Development

ABSTRACT The assembly of newly synthesized DNA into chromatin is essential for normal growth, development, and differentiation. To gain a better understanding of the assembly of chromatin during DNA synthesis, we identified, cloned, and characterized the 180- and 105-kDa polypeptides of Drosophila chromatin assembly factor 1 (dCAF-1). The purified recombinant p180+p105+p55 dCAF-1 complex is active for DNA replication-coupled chromatin assembly. Furthermore, we have established that the putative 75-kDa polypeptide of dCAF-1 is a C-terminally truncated form of p105 that does not coexist in dCAF-1 complexes containing the p105 subunit. The analysis of native and recombinant dCAF-1 revealed an interaction between dCAF-1 and theDrosophila anti-silencing function 1 (dASF1) component of replication-coupling assembly factor (RCAF). The binding of dASF1 to dCAF-1 is mediated through the p105 subunit of dCAF-1. Consistent with the interaction between dCAF-1 p105 and dASF1 in vitro, we observed that dASF1 and dCAF-1 p105 colocalized in vivo inDrosophila polytene chromosomes. This interaction between dCAF-1 and dASF1 may be a key component of the functional synergy observed between RCAF and dCAF-1 during the assembly of newly synthesized DNA into chromatin.

Synthesis of acid-soluble spore proteins by Bacillus subtilis

1982 ◽  
Vol 152 (3) ◽  
pp. 1117-1125
Author(s):  
J M Leventhal ◽  
G H Chambliss
Keyword(s):  
Bacillus Subtilis ◽  
Maximum Rate ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Sodium Dodecyl ◽  
Molecular Weights ◽  
Spore Proteins ◽  
Major Acid

The major acid-soluble spore proteins (ASSPs) of Bacillus subtilis were detected by immunoprecipitation of radioactively labeled in vitro- and in vivo-synthesized proteins. ASSP synthesis in vivo began 2 h after the initiation of sporulation (t2) and reached its maximum rate at t7. This corresponded to the time of synthesis of mRNA that stimulated the maximum rate of ASSP synthesis in vitro. Under the set of conditions used in these experiments, protease synthesis began near t0, alkaline phosphatase synthesis began at about t2, and refractile spores were first observed between t7 and t8. In vivo- and in vitro-synthesized ASSPs comigrated in sodium dodecyl sulfate-polyacrylamide gels. Their molecular weights were 4,600 (alpha and beta) and 11,000 (gamma). The average half-life of the ASSP messages was 11 min when either rifampin (10 micrograms/ml) or actinomycin D (1 microgram/ml) was used to inhibit RNA synthesis.

RNA binding by Sxl proteins in vitro and in vivo

1994 ◽  
Vol 14 (7) ◽  
pp. 4975-4990
Author(s):  
M E Samuels ◽  
D Bopp ◽  
R A Colvin ◽  
R F Roscigno ◽  
M A Garcia-Blanco ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Target Genes ◽  
Rna Binding ◽  
Polytene Chromosomes ◽  
Regulatory Sequences ◽  
Acceptor Site ◽  
Nuclear Extracts ◽  
Preferential Binding

Sxl has been proposed to regulate splicing of specific target genes by directly interacting with their pre-mRNAs. We have therefore examined the RNA-binding properties of Sxl protein in vitro and in vivo. Gel shift and UV cross-linking assays with a purified recombinant MBP-Sxl fusion protein demonstrated preferential binding to RNAs containing poly(U) tracts, and the protein footprinted over the poly(U) region. The protein did not appear to recognize either branch point or AG dinucleotide sequences, but an adenosine residue at the 5' end of the poly(U) tract enhanced binding severalfold. MBP-Sxl formed two shifted complexes on a tra regulated acceptor site RNA; the doubly shifted form may have been stabilized by protein-protein interactions. Consistent with its proposed role in pre-mRNA processing, in nuclear extracts Sxl was found in large ribonucleoprotein (RNP) complexes which sedimented significantly faster than bulk heterogeneous nuclear RNP and small nuclear RNPs. Anti-Sxl staining of polytene chromosomes showed Sxl protein at a number of chromosomal locations, among which was the Sxl locus itself. Sxl protein could also be targeted to a new chromosomal site carrying a transgene containing splicing regulatory sequences from the Sxl gene, following transcriptional induction. After prolonged heat shock, all Sxl protein was restricted to the heat-induced puff at the hs93D locus. In contrast, a presumptive small nuclear RNP protein was observed at several heat puffs following shock.

Cellular heterogeneity of uridine incorporation in collecting tubules: effect of DOCA

1985 ◽  
Vol 248 (4) ◽  
pp. F552-F564
Author(s):  
A. Vandewalle ◽  
F. Cluzeaud ◽  
M. Chavance ◽  
J. P. Bonvalet
Keyword(s):  
Rna Synthesis ◽  
Cellular Heterogeneity ◽  
Nuclear Surface ◽  
Intercalated Cells ◽  
Uridine Incorporation ◽  
Collecting Tubules ◽  
Silver Grains ◽  
Stimulation Of

In previous studies we showed that in vitro uridine incorporation along the renal tubule is heterogeneous and that DOCA induces a stimulation of RNA synthesis in distal cortical and medullary structures. The present work examines by autoradiography of isolated tubules and renal tissue sections the cellular heterogeneity of the connecting (CNT) and cortical collecting (CCT) tubules after in vivo injection of [3H]uridine in normal and DOCA-treated rabbits. Data confirmed the profile of uridine incorporation along the tubule, which was found in in vitro experiments, and the DOCA-induced stimulation of RNA synthesis. In microdissected CNT and CCT of control kidneys, statistical analysis of the distribution of labeling revealed the presence of two distinct cell populations: one with low labeling (2-3 silver grains per nucleus) and one with high labeling (10-13), which represent 64 and 36%, respectively (CNT), and 74 and 26%, respectively (CCT), of the whole population. Histological data showed that the respective proportions of intercalated cells (29% in CNT; 21% in CCT) and connecting tubule cells (65%) or principal cells (79%) are close to those of the populations with high or low labeling. In addition, autoradiographs on renal sections directly demonstrated that the labeling of intercalated cells (19.3 silver grains/100 micron2 nuclear surface in CNT; 14.7 in CCT) was three times higher than that of connecting (6.6) or principal (5.8) cells. In isolated CNT and CCT, DOCA induced similar absolute increases in the labeling of the two populations. However, the relative increase was more than two times higher in the population with low labeling (+131% in CNT, +210% in CCT) than in the one with high labeling (+71% and +98%). We conclude that cell population of the collecting cortical tubule (CNT and CCT) is heterogeneous with regard to uridine incorporation, reflecting RNA synthesis.

Ribonucleic acid synthesis inPlasmodium knowlesimaintained bothin vivoandin vitro

Parasitology ◽  
1975 ◽  
Vol 71 (2) ◽  
pp. 199-209 ◽  
Author(s):  
P. I. Trigg ◽  
P. G. Shakespeare ◽  
Susan J. Burt ◽  
Sally I. Kyd
Keyword(s):  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Nuclear Division ◽  
Plasmodium Knowlesi ◽  
Acid Synthesis ◽  
Agarose Gel Electrophoresis ◽  
Trophozoite Stage ◽  
Late Trophozoite

RNA extracted from purified parasites ofPlasmodium knowlesiwas fractionated using agarose gel electrophoresis. Preparations from parasites grown bothin vivoandin vitrocontained species of RNA with sedimentation coefficients of 4·0S, 5·0S, 16·6S, 24·2S, 31·4S, 38·0S and 48·3S. There was less RNA present in parasites grownin vitrothan the equivalent stage parasites grownin vivobut the proportional amounts of the various species of RNA was similar in both cases. It is suggested that the 24·2S and 16·6S species of RNA are ribosomal and that the high molecular weight 31·4S, 38·0S and 48·0S species are ribosomal precursors. Ribosomal RNA synthesis occurs throughout the cell cycle during growth from the ring to the schizont stage; maximum incorporation of [H3]-adenosine occurs at the late trophozoite stage before nuclear division.

scholarly journals Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA

2017 ◽  
Vol 115 (2) ◽  
pp. E162-E171 ◽  
Author(s):  
François Ferron ◽  
Lorenzo Subissi ◽  
Ana Theresa Silveira De Morais ◽  
Nhung Thi Tuyet Le ◽  
Marion Sevajol ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Mutation Rates ◽  
Bifunctional Enzyme ◽  
Antiviral Compound ◽  
Mismatch Correction ◽  
Limited Efficacy

Coronaviruses (CoVs) stand out among RNA viruses because of their unusually large genomes (∼30 kb) associated with low mutation rates. CoVs code for nsp14, a bifunctional enzyme carrying RNA cap guanine N7-methyltransferase (MTase) and 3′-5′ exoribonuclease (ExoN) activities. ExoN excises nucleotide mismatches at the RNA 3′-end in vitro, and its inactivation in vivo jeopardizes viral genetic stability. Here, we demonstrate for severe acute respiratory syndrome (SARS)-CoV an RNA synthesis and proofreading pathway through association of nsp14 with the low-fidelity nsp12 viral RNA polymerase. Through this pathway, the antiviral compound ribavirin 5′-monophosphate is significantly incorporated but also readily excised from RNA, which may explain its limited efficacy in vivo. The crystal structure at 3.38 Å resolution of SARS-CoV nsp14 in complex with its cofactor nsp10 adds to the uniqueness of CoVs among RNA viruses: The MTase domain presents a new fold that differs sharply from the canonical Rossmann fold.

Application of Escherichia coli phage K1E DNA-dependent RNA polymerase for in vitro RNA synthesis and in vivo protein production in Bacillus megaterium

2010 ◽  
Vol 88 (2) ◽  
pp. 529-539 ◽  
Author(s):  
Simon Stammen ◽  
Franziska Schuller ◽  
Sylvia Dietrich ◽  
Martin Gamer ◽  
Rebekka Biedendieck ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Bacillus Megaterium ◽  
Protein Production ◽  
Rna Synthesis ◽  
Escherichia Coli Phage
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