DIFFERENTIATION OF DROSOPHILA CELLS LACKING RIBOSOMAL DNA, IN VITRO

ABSTRACT Drosophila melanogaster embryos that lacked ribosomal DNA were obtained from appropriate crosses. Cells were taken from such embryos before overt differentiation took place and were cultured in vitro. These cells differentiated into neurons and myocytes with the same success as did wild-type controls. Therefore, ribosomal RNA synthesis is not necessary for the differentiation of neurons and myocytes in vitro.

Download Full-text

REGULATION OF RIBOSOMAL RNA AND 5s RNA SYNTHESIS IN DROSOPHILA MELANOGASTER: I. BOBBED MUTANTS

Genetics ◽

10.1093/genetics/72.2.267 ◽

1972 ◽

Vol 72 (2) ◽

pp. 267-276

Author(s):

Roberto Weinmann

Keyword(s):

Drosophila Melanogaster ◽

Developmental Delay ◽

Ribosomal Rna ◽

Gel Electrophoresis ◽

Rna Synthesis ◽

Rrna Synthesis ◽

Wild Type ◽

5S Rna ◽

Wild Type Level

ABSTRACT Analysis of the rates and amounts of rRNA and 5s RNA synthesized in Drosophila melanogaster bobbed mutants was done by using acrylamide-gel electrophoresis. The results show that the amounts of rRNA synthesized are constant, although the rates of rRNA synthesis in bb's are reduced to 30% of the wild-type level. The rates of synthesis of 5s RNA were constant. The rate of synthesis of the two kinds of molecules that enter in equimolar amounts into the mature ribosome is non-coordinated.—The rates of rRNA synthesis were shown to be proportional to the length of the scutellar bristles, supporting the notion that in trichogen cells there is no developmental delay, but the size of the bristle depends directly on the rate of rRNA synthesis.

Download Full-text

Synthesis of RNA in oocytes of Xenopus laevis during culture in vitro

Development ◽

10.1242/dev.32.2.515 ◽

1974 ◽

Vol 32 (2) ◽

pp. 515-532

Author(s):

A. Colman

Keyword(s):

Xenopus Laevis ◽

In Vitro Culture ◽

Ribosomal Rna ◽

Rna Synthesis ◽

Culture Medium ◽

Salt Medium ◽

Complex Media ◽

18S Ribosomal Rna ◽

Rna Precursor

RNA synthesis can be maintained in large oocytes of Xenopus laevis during periods of in vitro culture of at least 10 days. A simple salt medium, modified Barth's solution, is found to be as effective a culture medium for these oocytes as several other complex media. The newly synthesized RNA is characterized electrophoretically and shown to consist predominantly of ribosomal RNA precursor, 28S and 18S ribosomal RNA, and 4S RNA. The distribution of this RNA within the oocyte is detected autoradiographically, where it is found to be greatly concentrated over the nucleoli. No qualitative alterations in either of these parameters are found during culture, within the limits of sensitivity of the assay procedures.

Download Full-text

Juvenile hormone production by wild type and apterous 4 mutant Drosophila melanogaster corpora allata in vitro

Insect Neurochemistry and Neurophysiology · 1989 · ◽

10.1007/978-1-4612-4512-4_37 ◽

1990 ◽

pp. 333-336 ◽

Cited By ~ 1

Author(s):

M. Altaratz ◽

D. Segal ◽

D. S. Richard ◽

L. I. Gilbert ◽

S. W. Applebaum

Keyword(s):

Drosophila Melanogaster ◽

Juvenile Hormone ◽

Corpora Allata ◽

Wild Type ◽

Hormone Production

Download Full-text

Calreticulin Mediates Anesthetic Sensitivity in Drosophila melanogaster

Anesthesiology ◽

10.1097/00000542-200310000-00019 ◽

2003 ◽

Vol 99 (4) ◽

pp. 867-875 ◽

Cited By ~ 16

Author(s):

Sumiko Gamo ◽

Junya Tomida ◽

Katsuyuki Dodo ◽

Dai Keyakidani ◽

Hitoshi Matakatsu ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Double Mutant ◽

Developmental Stages ◽

Northern Blotting ◽

General Anesthetics ◽

Wild Type ◽

Mutant Strains ◽

Low Expression

Background Various species, e.g., Caenorhabditis elegans, Drosophila melanogaster, and mice, have been used to explore the mechanisms of action of general anesthetics in vivo. The authors isolated a Drosophila mutant, ethas311, that was hypersensitive to diethylether and characterized the calreticulin (crc) gene as a candidate of altered anesthetic sensitivity. Methods Molecular analysis of crc included cloning and sequencing of the cDNA, Northern blotting, and in situ hybridization to accomplish the function of the gene and its mutation. For anesthetic phenotype assay, the 50% anesthetizing concentrations were determined for ethas311, revertants, and double-mutant strains (wild-type crc transgene plus ethas311). Results Expression of the crc 1.4-kb transcript was lower in the mutant ethas311 than in the wild type at all developmental stages. The highest expression at 19 h after pupation was observed in the brain of the wild type but was still low in the mutant at that stage. The mutant showed resistance to isoflurane as well as hypersensitivity to diethylether, whereas it showed the wild phenotype to halothane. Both mutant phenotypes were restored to the wild type in the revertants and double-mutant strains. Conclusion ethas311 is a mutation of low expression of the Drosophila calreticulin gene. The authors demonstrated that hypersensitivity to diethylether and resistance to isoflurane are associated with low expression of the gene. In Drosophila, calreticulin seems to mediate these anesthetic sensitivities, and it is a possible target for diethylether and isoflurane, although the predicted anesthetic targets based on many studies in vitro and in vivo are the membrane proteins, such as ion channels and receptors.

Download Full-text

Cleavage between Replicase Proteins p28 and p65 of Mouse Hepatitis Virus Is Not Required for Virus Replication

Journal of Virology ◽

10.1128/jvi.78.11.5957-5965.2004 ◽

2004 ◽

Vol 78 (11) ◽

pp. 5957-5965 ◽

Cited By ~ 40

Author(s):

Mark R. Denison ◽

Boyd Yount ◽

Sarah M. Brockway ◽

Rachel L. Graham ◽

Amy C. Sims ◽

...

Keyword(s):

Rna Synthesis ◽

Hepatitis Virus ◽

Mouse Hepatitis Virus ◽

Wild Type ◽

Virus Growth ◽

Amino Terminal ◽

Normal Expression ◽

Infected Cells ◽

Critical Residues

ABSTRACT The p28 and p65 proteins of mouse hepatitis virus (MHV) are the most amino-terminal protein domains of the replicase polyprotein. Cleavage between p28 and p65 has been shown to occur in vitro at cleavage site 1 (CS1), 247Gly↓Val248, in the polyprotein. Although critical residues for CS1 cleavage have been mapped in vitro, the requirements for cleavage have not been studied in infected cells. To define the determinants of CS1 cleavage and the role of processing at this site during MHV replication, mutations and deletions were engineered in the replicase polyprotein at CS1. Mutations predicted to allow cleavage at CS1 yielded viable virus that grew to wild-type MHV titers and showed normal expression and processing of p28 and p65. Mutant viruses containing predicted noncleaving mutations or a CS1 deletion were also viable but demonstrated delayed growth kinetics, reduced peak titers, decreased RNA synthesis, and small plaques compared to wild-type controls. No p28 or p65 was detected in cells infected with predicted noncleaving CS1 mutants or the CS1 deletion mutant; however, a new protein of 93 kDa was detected. All introduced mutations and the deletion were retained during repeated virus passages in culture, and no phenotypic reversion was observed. The results of this study demonstrate that cleavage between p28 and p65 at CS1 is not required for MHV replication. However, proteolytic separation of p28 from p65 is necessary for optimal RNA synthesis and virus growth, suggesting important roles for these proteins in the formation or function of viral replication complexes.

Download Full-text

Ribonucleic acid synthesis inPlasmodium knowlesimaintained bothin vivoandin vitro

Parasitology ◽

10.1017/s0031182000046655 ◽

1975 ◽

Vol 71 (2) ◽

pp. 199-209 ◽

Cited By ~ 13

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.

Download Full-text

Formation of an active transcription complex in the Drosophila melanogaster 5S RNA gene is dependent on an upstream region.

Molecular and Cellular Biology ◽

10.1128/mcb.7.6.2046 ◽

1987 ◽

Vol 7 (6) ◽

pp. 2046-2051 ◽

Cited By ~ 18

Author(s):

A D Garcia ◽

A M O'Connell ◽

S J Sharp

Keyword(s):

Drosophila Melanogaster ◽

Transcription Initiation ◽

Rna Polymerase Iii ◽

In Vitro Transcription ◽

Upstream Region ◽

Nucleotide Position ◽

Wild Type ◽

5S Rna ◽

Cell Extracts

We constructed deletion-substitution and linker-scanning mutations in the 5'-flanking region of the Drosophila melanogaster 5S RNA gene. In vitro transcription of these templates in Drosophila and HeLa cell extracts revealed the presence of an essential control region (-30 region) located between nucleotides -39 and -26 upstream of the transcription initiation site: deletion of sequences upstream of nucleotide position -39 had no detectable effect on the wild-type level of in vitro transcription, whereas mutations extending between positions -39 and 1 resulted in templates with decreased transcriptional levels; specifically, deletion and linker-scanning mutations in the -34 to -26 region (-30 region) resulted in loss of transcription. The -30 region is essential for transcription and therefore forms part of the Drosophila 5S RNA gene transcription promoter. Compared with the activity of the wild-type gene, mutant 5S DNAs exhibited no impairment in the ability to sequester limiting transcription factors in a template exclusion competition assay. While we do not know which transcription factor(s) interacts with the -30 region, the possible involvement of RNA polymerase III at this region is discussed.

Download Full-text

Differential Rescue of Poliovirus RNA Replication Functions by Genetically Modified RNA Polymerase Precursors

Journal of Virology ◽

10.1128/jvi.78.23.13007-13018.2004 ◽

2004 ◽

Vol 78 (23) ◽

pp. 13007-13018 ◽

Cited By ~ 7

Author(s):

Christopher T. Cornell ◽

Jo Ellen Brunner ◽

Bert L. Semler

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Genetically Modified ◽

Rna Replication ◽

Amino Acid Sequences ◽

In Vitro Translation ◽

Wild Type ◽

Positive Strand ◽

Positive Strand Rna

ABSTRACT We have previously described the RNA replication properties of poliovirus transcripts harboring chimeric RNA polymerase sequences representing suballelic exchanges between poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3) utilizing an in vitro translation and RNA replication assay (C. Cornell, R. Perera, J. E. Brunner, and B. L. Semler, J. Virol. 78:4397-4407, 2004). We showed that three of the seven chimeras were capable of RNA replication in vitro, although replication levels were greatly reduced compared to that of wild-type transcripts. Interestingly, one of the replication-competent transcripts displayed a strand-specific RNA synthesis defect suggesting (i) a differential replication complex assembly mechanism involving 3D and/or precursor molecules (i.e., 3CD) required for negative- versus positive-strand RNA synthesis or (ii) effect(s) on the ability of the 3D polymerase to form higher-ordered structures required for positive-strand RNA synthesis. In this study, we have attempted to rescue defective RNA replication in vitro by cotranslating nonstructural proteins from a transcript encoding a large precursor polyprotein (P3) to complement 3D polymerase and/or precursor polypeptide functions altered in each of the chimeric constructs. Utilization of a wild-type P3 construct revealed that all transcripts containing chimeric PV1/CVB3 polymerase sequences can be complemented in trans for both negative- and positive-strand RNA synthesis. Furthermore, data from experiments utilizing genetically modified forms of the P3 polyprotein, containing mutations within 3C or 3D sequences, strongly suggest the existence of different protein-protein and protein-RNA interactions required for positive- versus negative-strand RNA synthesis. These results, combined with data from in vitro RNA elongation assays, indicate that the delivery of active 3D RNA polymerase to replication complexes requires a series of macromolecular interactions that rely on the presence of specific 3D amino acid sequences.

Download Full-text