Translational regulation of light-induced ribulose 1,5-bisphosphate carboxylase gene expression in amaranth

The regulation of the genes encoding the large and small subunits of ribulose 1,5-bisphosphate carboxylase was examined in amaranth cotyledons in response to changes in illumination. When dark-grown cotyledons were transferred into light, synthesis of the large- and small-subunit polypeptides was initiated very rapidly, before any increase in the levels of their corresponding mRNAs. Similarly, when light-grown cotyledons were transferred to total darkness, synthesis of the large- and small-subunit proteins was rapidly depressed without changes in mRNA levels for either subunit. In vitro translation or in vivo pulse-chase experiments indicated that these apparent changes in protein synthesis were not due to alterations in the functionality of the mRNAs or to protein turnover, respectively. These results, in combination with our previous studies, suggest that the expression of ribulose 1,5-bisphosphate carboxylase genes can be adjusted rapidly at the translational level and over a longer period through changes in mRNA accumulation.

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Transcriptional and post-transcriptional regulation of ribulose 1,5-bisphosphate carboxylase gene expression in light- and dark-grown amaranth cotyledons.

Molecular and Cellular Biology ◽

10.1128/mcb.5.9.2238 ◽

1985 ◽

Vol 5 (9) ◽

pp. 2238-2246 ◽

Cited By ~ 84

Author(s):

J O Berry ◽

B J Nikolau ◽

J P Carr ◽

D F Klessig

Keyword(s):

Specific Activity ◽

Large Subunit ◽

Growth Conditions ◽

Small Subunit ◽

Northern Analysis ◽

Mrna Levels ◽

Regulation Of Expression ◽

Bisphosphate Carboxylase

The regulation of expression of the genes encoding the large subunit (LSU) and small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase (RuBPCase) was examined in 1- through 8-day-old, dark-grown (etiolated) and light-grown amaranth cotyledons. RuBPCase specific activity in light-grown cotyledons increased during this 8-day period to a level 15-fold higher than in dark-grown cotyledons. Under both growth conditions, the accumulation of the LSU and SSU polypeptides was not coordinated. Initial detection of the SSU occurred 1 and 2 days after the appearance of the LSU in light- and dark-grown cotyledons, respectively. Furthermore, although the levels of the LSU were similar in both light- and dark-grown seedlings, the amount of the SSU followed clearly the changes in enzyme activity. Synthesis of these two polypeptides was dramatically different in etiolated versus light-grown cotyledons. In light the synthesis of both subunits was first observed on day 2 and continued throughout the growth of the cotyledons. In darkness the rate of synthesis of both subunits was much lower than in light and occurred only as a burst between days 2 and 5 after planting. However, mRNAs for both subunits were present in etiolated cotyledons at similar levels on days 4 through 7 (by Northern analysis) and were functional in vitro, despite their apparent inactivity in vivo after day 5. In addition, since both LSU and SSU mRNA levels were lower in dark- than in light-grown seedlings, our results indicate that both transcriptional and post-transcriptional controls modulate RuBPCase production in developing amaranth cotyledons.

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Transcriptional and post-transcriptional regulation of ribulose 1,5-bisphosphate carboxylase gene expression in light- and dark-grown amaranth cotyledons

Molecular and Cellular Biology ◽

10.1128/mcb.5.9.2238-2246.1985 ◽

1985 ◽

Vol 5 (9) ◽

pp. 2238-2246

Author(s):

J O Berry ◽

B J Nikolau ◽

J P Carr ◽

D F Klessig

Keyword(s):

Specific Activity ◽

Large Subunit ◽

Growth Conditions ◽

Small Subunit ◽

Northern Analysis ◽

Mrna Levels ◽

Regulation Of Expression ◽

Bisphosphate Carboxylase

The regulation of expression of the genes encoding the large subunit (LSU) and small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase (RuBPCase) was examined in 1- through 8-day-old, dark-grown (etiolated) and light-grown amaranth cotyledons. RuBPCase specific activity in light-grown cotyledons increased during this 8-day period to a level 15-fold higher than in dark-grown cotyledons. Under both growth conditions, the accumulation of the LSU and SSU polypeptides was not coordinated. Initial detection of the SSU occurred 1 and 2 days after the appearance of the LSU in light- and dark-grown cotyledons, respectively. Furthermore, although the levels of the LSU were similar in both light- and dark-grown seedlings, the amount of the SSU followed clearly the changes in enzyme activity. Synthesis of these two polypeptides was dramatically different in etiolated versus light-grown cotyledons. In light the synthesis of both subunits was first observed on day 2 and continued throughout the growth of the cotyledons. In darkness the rate of synthesis of both subunits was much lower than in light and occurred only as a burst between days 2 and 5 after planting. However, mRNAs for both subunits were present in etiolated cotyledons at similar levels on days 4 through 7 (by Northern analysis) and were functional in vitro, despite their apparent inactivity in vivo after day 5. In addition, since both LSU and SSU mRNA levels were lower in dark- than in light-grown seedlings, our results indicate that both transcriptional and post-transcriptional controls modulate RuBPCase production in developing amaranth cotyledons.

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Sheep antiluteolytic interferon: cDNA sequence and analysis of mRNA levels

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0020065 ◽

1989 ◽

Vol 2 (1) ◽

pp. 65-70 ◽

Cited By ~ 40

Author(s):

H.J. Stewart ◽

S.H.E. McCann ◽

A.J. Northrop ◽

G.E. Lamming ◽

A.P.F. Flint

Keyword(s):

Amino Acid ◽

Northern Blotting ◽

In Vitro Translation ◽

Major Constituent ◽

Interferon Α ◽

Mrna Levels ◽

Blastocyst Development ◽

Cdna Sequences

ABSTRACT A cloned cDNA has been isolated by probing a sheep blastocyst cDNA library using a synthetic oligonucleotide representing the N-terminal amino acid sequence of the antiluteolytic protein, ovine trophoblast protein-1. Sequence analysis of the cDNA confirms the 70% homology between the antiluteolysin and the interferon-α family of proteins; however, the sequence reported here differs at several points from previously reported amino acid and cDNA sequences for the antiluteolysin. In-vitro translation of day-16 poly(A)+ RNA indicated that antiluteolysin mRNA is a major constituent of total mRNA at this stage of blastocyst development, and Northern blotting confirmed that antiluteolysin mRNA production occurred between days 13 and 22 after oestrus. This is consistent with the stage at which embryonic extracts are antiluteolytic on administration in vivo. These and other data confirm that the ovine trophoblast antiluteolysin is an interferon, and suggest that at least five isoforms of this protein may exist.

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c-erbA encodes multiple proteins in chicken erythroid cells.

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4155 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4155-4161 ◽

Cited By ~ 36

Author(s):

J Bigler ◽

R N Eisenman

Keyword(s):

Expression Vector ◽

In Vitro Translation ◽

Vector System ◽

Mrna Levels ◽

Erythroid Cells ◽

Avian Erythroblastosis Virus ◽

Related Proteins ◽

Translational Mechanisms

To identify and characterize the proteins encoded by the erbA proto-oncogene, we expressed the C-terminal region of v-erbA in a bacterial trpE expression vector system and used the fusion protein to prepare antiserum. The anti-trp-erbA serum recognized the P75gag-erbA protein encoded by avian erythroblastosis virus and specifically precipitated six highly related proteins ranging in size from 27 to 46 kilodaltons from chicken embryonic erythroid cells. In vitro translation of a chicken erbA cDNA produced essentially the same pattern of proteins. Partial proteolytic maps and antigenicity and kinetic analyses of the in vivo and in vitro proteins indicated that they are related and that the multiple bands are likely to arise from internal initiations within c-erbA to generate a nested set of proteins. All of the c-erbA proteins are predominantly associated with chicken erythroblast nuclei. However, Nonidet P-40 treatment resulted in extraction of the three smaller proteins, whereas the larger proteins were retained. During differentiation of erythroid cells in chicken embryos, we found maximal levels of c-erbA protein synthesis at days 7 to 8 of embryogenesis. By contrast, c-erbA mRNA levels remained essentially constant from days 5 to 12. Together, our results indicate that posttranscriptional or translational mechanisms are involved in regulation of c-erbA expression and in the complexity of its protein products.

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c-erbA encodes multiple proteins in chicken erythroid cells

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4155-4161.1988 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4155-4161

Author(s):

J Bigler ◽

R N Eisenman

Keyword(s):

Expression Vector ◽

In Vitro Translation ◽

Vector System ◽

Mrna Levels ◽

Erythroid Cells ◽

Avian Erythroblastosis Virus ◽

Related Proteins ◽

Translational Mechanisms

To identify and characterize the proteins encoded by the erbA proto-oncogene, we expressed the C-terminal region of v-erbA in a bacterial trpE expression vector system and used the fusion protein to prepare antiserum. The anti-trp-erbA serum recognized the P75gag-erbA protein encoded by avian erythroblastosis virus and specifically precipitated six highly related proteins ranging in size from 27 to 46 kilodaltons from chicken embryonic erythroid cells. In vitro translation of a chicken erbA cDNA produced essentially the same pattern of proteins. Partial proteolytic maps and antigenicity and kinetic analyses of the in vivo and in vitro proteins indicated that they are related and that the multiple bands are likely to arise from internal initiations within c-erbA to generate a nested set of proteins. All of the c-erbA proteins are predominantly associated with chicken erythroblast nuclei. However, Nonidet P-40 treatment resulted in extraction of the three smaller proteins, whereas the larger proteins were retained. During differentiation of erythroid cells in chicken embryos, we found maximal levels of c-erbA protein synthesis at days 7 to 8 of embryogenesis. By contrast, c-erbA mRNA levels remained essentially constant from days 5 to 12. Together, our results indicate that posttranscriptional or translational mechanisms are involved in regulation of c-erbA expression and in the complexity of its protein products.

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Degradation of the soybean ribulose-1,5-bisphosphate carboxylase small-subunit mRNA, SRS4, initiates with endonucleolytic cleavage.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.6641 ◽

1995 ◽

Vol 15 (12) ◽

pp. 6641-6652 ◽

Cited By ~ 14

Author(s):

M M Tanzer ◽

R B Meagher

Keyword(s):

Consensus Sequence ◽

Random Order ◽

Initial Step ◽

Preliminary Evidence ◽

Small Subunit ◽

S1 Nuclease ◽

Bisphosphate Carboxylase ◽

Endonucleolytic Cleavage

The degradation of the soybean SRS4 mRNA, which encodes the small subunit of ribulose-1,5-bisphosphate carboxylase, yields a set of proximal (5' intact) and distal (3' intact) products both in vivo and in vitro. These products are generated by endonucleolytic cleavages that occur essentially in a random order, although some products are produced more rapidly than others. Comparison of sizes of products on Northern (RNA) blots showed that the combined sizes of pairs of proximal and distal products form contiguous full-length SRS4 mRNAs. When the 3' ends of the proximal products and the 5' ends of the distal products were mapped by S1 nuclease and primer extension assays, respectively, both sets of ends mapped to the same sequences within the SRS4 mRNA. A small in vitro-synthesized RNA fragment containing one cleavage site inhibited cleavage of all major sites, equivalently consistent with one enzymatic activity generating the endonucleolytic cleavage products. These products were rich in GU nucleotides, but no obvious consensus sequence was found among several cleavage sites. Preliminary evidence suggested that secondary structure could play a role in site selection. The structures of the 5' ends of the proximal products and the 3' ends of the distal products were examined. Proximal products were found with approximately equal frequency in both m7G cap(+) and m7G cap(-) fractions, suggesting that the endonucleolytic cleavage events occurred independently of the removal of the 5' cap structure. Distal products were distributed among fractions with poly(A) tails ranging from undetectable to greater than 100 nucleotides in length, suggesting that the endonucleolytic cleavage events occurred independently of poly(A) tail shortening. Together, these data support a stochastic endonuclease model in which an endonucleolytic cleavage event is the initial step in SRS4 mRNA degradation.

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Isolation of the human genes encoding the pyst1 and Pyst2 phosphatases: characterisation of Pyst2 as a cytosolic dual-specificity MAP kinase phosphatase and its catalytic activation by both MAP and SAP kinases

Journal of Cell Science ◽

10.1242/jcs.111.22.3389 ◽

1998 ◽

Vol 111 (22) ◽

pp. 3389-3399 ◽

Cited By ~ 2

Author(s):

S. Dowd ◽

A.A. Sneddon ◽

S.M. Keyse

Keyword(s):

Map Kinase ◽

Substrate Selectivity ◽

Mrna Levels ◽

Catalytic Activation ◽

Dual Specificity ◽

Human Genes ◽

Map Kinase Phosphatase ◽

Genes Encoding

We have isolated the human genes encoding the Pyst1 (MKP-3) and Pyst2 (MKP-X) MAP kinase phosphatases. Both genes consist of three exons interrupted by two introns and lack an intron which is conserved in all the other members of this gene family characterised to date. This reinforces the conclusion that Pyst1 and Pyst2 are members of a distinct and structurally homologous subfamily of dual-specificity (Thr/Tyr) MAP kinase phosphatases. We find that Pyst2 mRNA is constitutively expressed in a wide variety of human cell lines including those derived from ovarian, bladder and breast cancers. While there is no evidence for inducible expression of Pyst2 mRNA in human skin fibroblasts in response to cellular stress, Pyst2 mRNA levels are moderately increased in response to serum stimulation. Pyst2 protein is predominantly cytosolic when expressed in COS-1 cells. In common with Pyst1, Pyst2 shows substrate selectivity for the classical p42 (ERK2) isoform of MAP kinase both in vitro and in vivo, displaying much reduced activity towards stress activated MAP kinase isoforms such as JNK-1 and p38/RK. Pyst2 binds p42 MAP kinase in vivo and both MAP kinase binding and substrate selectivity correlate with the ability of different recombinant MAP and SAP kinases to cause catalytic activation of the Pyst2 phosphatase in vitro.

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Cdk8 directly regulates glycolysis via phosphorylation of Gcr2

10.1101/2021.03.12.435012 ◽

2021 ◽

Author(s):

Maria J. Aristizabal ◽

Eoghan O’Duibhir ◽

Wim de Jonge ◽

Kristy Dever ◽

Nicole Hawe ◽

...

Keyword(s):

Gene Expression ◽

Transcription Regulation ◽

Time Course ◽

Phosphorylation Site ◽

Mrna Levels ◽

Functional Link ◽

Genes Encoding ◽

Context Specific

AbstractCDK8encodes an evolutionarily conserved Mediator complex kinase subunit that functions in general and context-specific transcription regulation by phosphorylating core components of the transcription machinery and gene-specific transcription factors. To better understand the role Cdk8 in transcription regulation, we performed high-resolution gene expression time course analysis following nuclear depletion of Cdk8. Focusing on the earliest gene expression alterations revealed dysregulation of genes encoding glycolysis enzymes, suggesting a functional link to Gcr1 and Gcr2, key transcriptional activators of these genes. Consistently, we found that nuclear depletion of Cdk8 altered the mRNA levels of glycolysis genes as well as the promoter occupancy of Gcr2, but not Gcr1. Examination of the Gcr2 protein sequence revealed a putative Cdk8 phosphorylation site at serine 365, which we confirmed usingin vitroandin vivoassays. Importantly, phospho-mutantGCR2recapitulated the growth and gene expression defects of theGCR2deletion mutant, effects not observed with a phospho mimetic mutant. As such, our work highlights Gcr2 as a new Cdk8 substrate, revealing that its phosphorylation is critical for the activation of genes encoding glycolysis enzymes.

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pp60c-src expression in transdifferentiating cultures of embryonic chick neural retina cells

Development ◽

10.1242/dev.101.4.847 ◽

1987 ◽

Vol 101 (4) ◽

pp. 847-856

Author(s):

D.K. Ellis ◽

A. Carr ◽

D.I. de Pomerai

Keyword(s):

Kinase Activity ◽

Neural Retina ◽

Northern Blotting ◽

In Vitro Translation ◽

Immunocytochemical Staining ◽

Mrna Levels ◽

Lens Cells ◽

Cell Groups

Chick embryo neural retinal cells transdifferentiate extensively into lens cells when cultured in Eagle's MEM containing horse and fetal calf sera (FHMEM). Such cultures express elevated levels of pp60c-src-associated tyrosine kinase activity relative to parallel cultures prevented from transdifferentiating by the addition of supplementary glucose (FHGMEM) or replacement of MEM by medium 199 (F199). Northern blotting and in vitro translation studies suggest that c-src mRNA levels are only slightly higher in late transdifferentiating (FHMEM) cultures as compared to parallel blocked (FHGMEM or F199) cultures. By immunocytochemical staining, we show that pp60c-src protein is largely localized in cell groups undergoing conversion into lens (i.e. expressing delta crystallin) in late FHMEM cultures. Initial studies of pp60c-src in chick lens tissues during development indicate that higher kinase activity is found in the epithelial cells relative to mature lens fibres. Thus pp60c-src may be expressed both during the differentiation of lens cells in vivo and during the transdifferentiation of neural retina cells into lens in vitro.

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