THE MEASLES VIRUS-SPECIFIC PROTEIN SYNTHESIS OF PERSISTENTLY AND LYTICALLY INFECTED CELLS STUDIED IN VIVO AND IN VITRO

ABSTRACT Pigmentation of the scrotum of the black-pelted rat, as expressed through melanocyte melanogenic activity, is controlled by androgens. Castration decreased in vitro incorporation of [14C] tyrosine into melanin. Testosterone pre-treatment for 4 days increased melanin radioactivity over castrate controls; the increment in vitro was prevented by an inhibitor of protein synthesis (cycloheximide) added to the incubation. However, cycloheximide only partially blocked melanin synthesis when added to tissue from animals hormone treated for 6 days in vivo, and was ineffective in tissue from intacts. Bulk protein synthesis in vitro (incorporation of [14C] tyrosine or -leucine) was not affected by castration or testosterone treatment but was uniformly inhibited by cycloheximide. The data suggest that new synthesis of specific protein in vitro was necessary for initial hormone-stimulation of melanogenesis, but with longer exposure to hormone sufficient protein was pre-synthetized in vivo to permit melanogenesis during incubation with the inhibitor. Radioautographs of epidermis incubated with [14C] tyrosine showed grains concentrated over macromolecular aggregates in melanocytes, a pattern not altered by cycloheximide. Though available for incorporation into general tissue protein. [14C] tyrosine was apparently incorporated preferentially into melanin by melanocytes. DOPA (3,4-dihydroxyphenylalanine) added to incubations in cofactor amounts did not affect decreased melanin synthesis after castration and appears, therefore, not to be rate limiting in that decrease. Tissue uptake of free [14C] tyrosine or — leucine during incubation was lower than normal in castrate epidermis; uptake was elevated by testosterone treatment. Concentrations appeared sufficient in all preparations to suggest that availability is not rate limiting for synthesis of melanin or protein; however, a possible influence on amino acid permeability for melanocytes remains undetermined. Tyrosinase activity was present in both particulate and cytosol fractions of epidermis but decreased significantly after castration only in the cytosol. Testosterone increased particulate activity after 4 days and soluble activity after 9 days of treatment. These and findings above are consistent with a model that tyrosinase is synthesized and incorporated into melanosome structure within 4 days testosterone treatment; with longer treatment synthesis may then exceed that required for melanosome assembly and tyrosinase appears in the soluble milieu.

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SPECIFIC PROTEIN SYNTHESIS IN CELLULAR DIFFERENTIATION

The Journal of Cell Biology ◽

10.1083/jcb.55.3.653 ◽

1972 ◽

Vol 55 (3) ◽

pp. 653-680 ◽

Cited By ~ 80

Author(s):

M. Paul ◽

M. R. Goldsmith ◽

J. R. Hunsley ◽

F. C. Kafatos

Keyword(s):

Protein Synthesis ◽

Messenger Rna ◽

Developmental Stages ◽

Specific Protein ◽

Chain Elongation ◽

Synthetic Activity ◽

Translational Efficiency ◽

Kinetics Of

Silkmoth follicles, arranged in a precise developmental sequence within the ovariole, yield pure and uniform populations of follicular epithelial cells highly differentiated for synthesis of the proteinaceous eggshell (chorion). These cells can be maintained and labeled efficiently in organ culture; their in vitro (and cell free) protein synthetic activity reflects their activity in vivo. During differentiation the cells undergo dramatic changes in protein synthesis. For 2 days the cells are devoted almost exclusively to production of distinctive chorion proteins of low molecular weight and of unusual amino acid composition. Each protein has its own characteristic developmental kinetics of synthesis. Each is synthesized as a separate polypeptide, apparently on monocistronic messenger RNA (mRNA), and thus reflects the expression of a distinct gene. The rapid changes in this tissue do not result from corresponding changes in translational efficiency. Thus, the peptide chain elongation rate is comparable for chorion and for proteins synthesized at earlier developmental stages (1.3–1.9 amino acids/sec); moreover, the spacing of ribosomes on chorion mRNA (30–37 codons per ribosome) is similar to that encountered in other eukaryotic systems.

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Characterization of Measles Virus-Specific Proteins Synthesized In Vivo and In Vitro from Acutely and Persistently Infected Cells

Journal of Virology ◽

10.1128/jvi.29.3.1099-1106.1979 ◽

1979 ◽

Vol 29 (3) ◽

pp. 1099-1106 ◽

Cited By ~ 10

Author(s):

Shmuel Rozenblatt ◽

Marian Gorecki ◽

Helen Shure ◽

Carol L. Prives

Keyword(s):

Measles Virus ◽

Persistently Infected ◽

Specific Proteins ◽

Infected Cells

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Specific Labeling of Protein Domains with Antibody Fragments

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100098587 ◽

1981 ◽

Vol 39 ◽

pp. 416-417

Author(s):

U. Aebi ◽

L.E. Buhle ◽

W.E. Fowler

Keyword(s):

Image Processing ◽

Specific Protein ◽

Antibody Fragments ◽

Supramolecular Organization ◽

Two Dimensional ◽

Ordered Structures ◽

Virus Capsids ◽

Processing Techniques

Many important supramolecular structures such as filaments, microtubules, virus capsids and certain membrane proteins and bacterial cell walls exist as ordered polymers or two-dimensional crystalline arrays in vivo. In several instances it has been possible to induce soluble proteins to form ordered polymers or two-dimensional crystalline arrays in vitro. In both cases a combination of electron microscopy of negatively stained specimens with analog or digital image processing techniques has proven extremely useful for elucidating the molecular and supramolecular organization of the constituent proteins. However from the reconstructed stain exclusion patterns it is often difficult to identify distinct stain excluding regions with specific protein subunits. To this end it has been demonstrated that in some cases this ambiguity can be resolved by a combination of stoichiometric labeling of the ordered structures with subunit-specific antibody fragments (e.g. Fab) and image processing of the electron micrographs recorded from labeled and unlabeled structures.

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Effect of microbial isolates on microbial yield estimation in RUSITEC system

BSAP Occasional Publication ◽

10.1017/s0263967x0003295x ◽

1998 ◽

Vol 22 ◽

pp. 306-308

Author(s):

M. D. Carro ◽

E. L. Miller

Keyword(s):

Protein Synthesis ◽

Liquid Phase ◽

Solid Phase ◽

Liquid Fraction ◽

Microbial Protein ◽

Microbial Protein Synthesis ◽

Continuous Culture System ◽

The One

The estimation of rumen microbial protein synthesis is one of the main points in the nitrogen (N)-rationing systems for ruminants, as microbial protein provides proportionately 0.4 to 0.9 of amino acids entering the small intestine in ruminants receiving conventional diets (Russell et al., 1992). Methods of estimating microbial protein synthesis rely on marker techniques in which a particular microbial constituent is related to the microbial N content. Marker : N values have generally been established in mixed bacteria isolated from the liquid fraction of rumen digesta and it has been assumed that the same relationship holds in the total population leaving the rumen (Merry and McAllan, 1983). However, several studies have demonstrated differences in composition between solid-associated (SAB) and fluid-associated bacteria in vivo (Legay-Carmier and Bauchart, 1989) and in vitro (Molina Alcaide et al, 1996), as well in marker : N values (Pérez et al., 1996). This problem could be more pronounced in the in vitro semi-continuous culture system RUSITEC, in which there are three well defined components (a free liquid phase, a liquid phase associated with the solid phase and a solid phase), each one having associated microbial populations.The objective of this experiment was to investigate the effect of using different bacterial isolates (BI) on the estimation of microbial production of four different diets in RUSITEC (Czerkawski and Breckenridge, 1977), using (15NH4)2 SO4 as microbial marker, and to assess what effects any differences would have on the comparison of microbial protein synthesis between diets.This study was conducted in conjunction with an in vitro experiment described by Carro and Miller (1997). Two 14-day incubation trials were carried out with the rumen simulation technique RUSITEC (Czerkawski and Breckenridge, 1977). The general incubation procedure was the one described by Czerkawski and Breckenridge (1977) and more details about the procedures of this experiment are given elsewhere (Carro and Miller, 1997).

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Studies of two temperature-sensitive mutants of Mengo virus

Canadian Journal of Biochemistry ◽

10.1139/o79-110 ◽

1979 ◽

Vol 57 (6) ◽

pp. 902-913 ◽

Cited By ~ 1

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.

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Phosphorylation of eukaryotic initiation factor 4E (eIF4E) at Ser209 is not required for protein synthesis in vitro and in vivo

European Journal of Biochemistry ◽

10.1046/j.0014-2956.2001.02478.x ◽

2001 ◽

Vol 268 (20) ◽

pp. 5375-5385 ◽

Cited By ~ 51

Author(s):

Linda McKendrick ◽

Simon J. Morley ◽

Virginia M. Pain ◽

Rosemary Jagus ◽

Bhavesh Joshi

Keyword(s):

Protein Synthesis ◽

Initiation Factor ◽

Eukaryotic Initiation Factor ◽

Eukaryotic Initiation Factor 4E

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Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1603271113 ◽

2016 ◽

Vol 113 (21) ◽

pp. E2899-E2905 ◽

Cited By ~ 23

Author(s):

Irina O. Vvedenskaya ◽

Hanif Vahedian-Movahed ◽

Yuanchao Zhang ◽

Deanne M. Taylor ◽

Richard H. Ebright ◽

...

Keyword(s):

Rna Polymerase ◽

Transcription Start Site ◽

Transcription Initiation ◽

Specific Protein ◽

Start Site ◽

Transcription Start ◽

Transcription Bubble ◽

Recognition Element

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the transcription bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

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