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

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

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2347-2353.1986 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2347-2353

Author(s):

J O Berry ◽

B J Nikolau ◽

J P Carr ◽

D F Klessig

Keyword(s):

Translational Regulation ◽

Small Subunit ◽

In Vitro Translation ◽

Mrna Levels ◽

Mrna Accumulation ◽

Bisphosphate Carboxylase ◽

Genes Encoding ◽

Translational Level

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

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2347 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2347-2353 ◽

Cited By ~ 56

Author(s):

J O Berry ◽

B J Nikolau ◽

J P Carr ◽

D F Klessig

Keyword(s):

Translational Regulation ◽

Small Subunit ◽

In Vitro Translation ◽

Mrna Levels ◽

Mrna Accumulation ◽

Bisphosphate Carboxylase ◽

Genes Encoding ◽

Translational Level

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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Investigation of in Vivo Roles of the C-terminal Tails of the Small Subunit (ββ′) of Saccharomyces cerevisiae Ribonucleotide Reductase

Journal of Biological Chemistry ◽

10.1074/jbc.m113.467001 ◽

2013 ◽

Vol 288 (20) ◽

pp. 13951-13959 ◽

Cited By ~ 9

Author(s):

Yan Zhang ◽

Xiuxiang An ◽

JoAnne Stubbe ◽

Mingxia Huang

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribonucleotide Reductase ◽

Large Subunit ◽

Small Subunit ◽

Cluster Assembly ◽

E Coli ◽

Viability Assay ◽

Docking Model

The small subunit (β2) of class Ia ribonucleotide reductase (RNR) houses a diferric tyrosyl cofactor (Fe2III-Y•) that initiates nucleotide reduction in the large subunit (α2) via a long range radical transfer (RT) pathway in the holo-(α2)m(β2)n complex. The C-terminal tails of β2 are predominantly responsible for interaction with α2, with a conserved tyrosine residue in the tail (Tyr356 in Escherichia coli NrdB) proposed to participate in cofactor assembly/maintenance and in RT. In the absence of structure of any holo-RNR, the role of the β tail in cluster assembly/maintenance and its predisposition within the holo-complex have remained unknown. In this study, we have taken advantage of the unusual heterodimeric nature of the Saccharomyces cerevisiae RNR small subunit (ββ′), of which only β contains a cofactor, to address both of these issues. We demonstrate that neither β-Tyr376 nor β′-Tyr323 (Tyr356 equivalent in NrdB) is required for cofactor assembly in vivo, in contrast to the previously proposed mechanism for E. coli cofactor maintenance and assembly in vitro. Furthermore, studies with reconstituted-ββ′ and an in vivo viability assay show that β-Tyr376 is essential for RT, whereas Tyr323 in β′ is not. Although the C-terminal tail of β′ is dispensable for cofactor formation and RT, it is essential for interactions with β and α to form the active holo-RNR. Together the results provide the first evidence of a directed orientation of the β and β′ C-terminal tails relative to α within the holoenzyme consistent with a docking model of the two subunits and argue against RT across the β β′ interface.

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Regulation of expression of mRNAs encoding the nerve growth factor receptors p75 and trkA in developing sensory neurons

Development ◽

10.1242/dev.119.3.635 ◽

1993 ◽

Vol 119 (3) ◽

pp. 635-648 ◽

Cited By ~ 17

Author(s):

S. Wyatt ◽

A.M. Davies

Keyword(s):

Neuronal Development ◽

Polymerase Chain Reaction Amplification ◽

Mrna Levels ◽

Regulation Of Expression ◽

Trigeminal Neurons ◽

Nerve Growth Factor Receptors ◽

Reaction Amplification ◽

Amplification Technique

We have used a quantitative reverse transcription/polymerase chain reaction amplification technique to study the regulation of p75 mRNA and trkA mRNA expression in developing NGF-dependent trigeminal neurons. Before becoming NGF dependent, these neurons express low levels of p75 and trkA mRNAs in vivo. At this stage in vitro, the level of p75 mRNA is maintained and up-regulated by BDNF, whereas the level of trkA mRNA is sustained independently of neurotrophins and is down-regulated by BDNF. With the acquisition of NGF dependence, p75 and trkA mRNA levels increase markedly in vivo. At this stage in vitro, the level of p75 mRNA is up-regulated by NGF, but this response is lost at later stages. The level of trkA mRNA is sustained in neurons grown with NGF but is not up-regulated by concentrations of NGF above those required to support survival. At no stage during the early development of trigeminal neurons do depolarising levels of potassium ions affect the expression of either p75 mRNA or trkA mRNA. These findings suggest that the expression of p75 and trkA mRNAs are differentially regulated by BDNF and NGF at successive early stages of neuronal development.

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Abstract 1190: The Complement Component C5a is Present in Human Unstable Coronary Lesions in Vivo and Induces the Expression of MMP-9 in Human Macrophages via Nf-kappaB Activation In Vitro

Circulation ◽

10.1161/circ.116.suppl_16.ii_241-b ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Walter S Speidl ◽

Stefan P Kastl ◽

Randolp Hutter ◽

Christoph Kaun ◽

Gerhard Bauriedel ◽

...

Keyword(s):

Specific Activity ◽

Complement Component ◽

Complement Cascade ◽

Mrna Levels ◽

Double Labeling ◽

Human Macrophages ◽

Coronary Lesions ◽

Coronary Events

Background. The complement component C5a formed during activation of the complement cascade exerts chemotactic and proinflammatory effects. We have recently shown that serum levels of C5a predict cardiovascular risk in patients with advanced atherosclerosis. However, it is not known whether C5a may play an active role in the onset of acute coronary events. Methods and Results. Immunohistochemical analysis of human coronary plaques obtained by atherectomy demonstrated the presence of C5a in macrophage rich areas and the necrotic core. Unstable plaques (n=9) showed significantly higher staining for C5a than stable coronary lesions (n=10; p<0.05). Double-labeling studies demonstrated colocalization of C5a and MMP-9. When human monocyte derived macrophages were stimulated in vitro with rhC5a, MMP-9 mRNA levels were increase 6-fold. In addition, MMP-9 antigen as detected by ELISA and MMP-9 activity as detected by a specific activity assay significantly increased after stimulation with rhC5a. Nuclear shift assay revealed that rhC5a stimulated NF-κB DNA binding and the NF-κB inhibitor 6-amino-4-phenoxyphenethylaminoquinazoline blocked the up-regulation of MMP-9 by rhC5a. Conclusion. C5a is increased in unstable coronary plaques, colocalizes with MMP-9 and induces the expression of MMP-9 in human macrophages via NF-κB activation. Taken this observations together, activation of the complement cascade and formation of C5a may be involved in the onset of acute coronary events.

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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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Retinoic Acid Inhibits Monocyte to Macrophage Survival and Differentiation

Blood ◽

10.1182/blood.v91.12.4796 ◽

1998 ◽

Vol 91 (12) ◽

pp. 4796-4802 ◽

Cited By ~ 26

Author(s):

Marina Kreutz ◽

Jana Fritsche ◽

Ute Ackermann ◽

Stefan W. Krause ◽

Reinhard Andreesen

Keyword(s):

Retinoic Acid ◽

Vitamin A ◽

Leukemic Cells ◽

Northern Analysis ◽

Enzyme Linked Immunosorbent Assay ◽

Mrna Levels ◽

Blood Monocytes ◽

Csf Production

Abstract Vitamin A metabolites are potent differentiation-inducing agents for myelomonocytic cell lines in vitro and are successfully used for the treatment of patients with acute promyelocytic leukemia. However, little is known about the effects of vitamin A on normal hematopoietic cells. Therefore, we investigated the effect of vitamin A on differentiation and activation of human blood monocytes (MO). Culturing MO for up to 4 days with 9-cis retinoic acid (RA) and all-trans RA but not retinol reduced MO survival, with the remaining cells being morphologically comparable to control cells. Because macrophage colony-stimulating factor (M-CSF) is a well-known survival factor for MO, we measured the M-CSF content of MO culture supernatants using enzyme-linked immunosorbent assay and found that RA suppressed the constitutive secretion of M-CSF. Northern analysis showed that the M-CSF mRNA expression was only slightly reduced by RA treatment, suggesting regulation on the posttranscriptional level. In contrast to MO, M-CSF secretion by MO-derived macrophages (MAC) was not altered by RA, suggesting a differentiation-dependent switch in the responsiveness of MO/MAC to RA. Because M-CSF is not only a survival-promoting but also a differentiation-promoting factor for myeloid cells, we analyzed the effect of RA on MO to MAC maturation. RA suppressed the expression of the maturation-associated antigen carboxypeptidase M (CPM)/MAX.1 at both the protein and mRNA levels and modulated the lipopolysaccharide-stimulated cytokine secretion of MO/MAC. The addition of exogenous M-CSF to RA-containing MO cultures fails to overcome the RA-induced inhibition of MO differentiation. However, the survival rate was improved by exogenous M-CSF. We conclude that RA acts via two different mechanisms on monocyte survival and differentiation: posttranscriptionally by controlling M-CSF secretion, which decreases MO survival, and transcriptionally regulating the expression of differentiation-associated genes. The regulation of M-CSF production may contribute to the antileukemic effect of RA in vivo by reducing autocrine M-CSF production by leukemic cells.

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Interaction between Subunits of Heterodimeric Splicing Factor U2AF Is Essential In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.18.4.1765 ◽

1998 ◽

Vol 18 (4) ◽

pp. 1765-1773 ◽

Cited By ~ 35

Author(s):

David Z. Rudner ◽

Roland Kanaar ◽

Kevin S. Breger ◽

Donald C. Rio

Keyword(s):

Amino Acid ◽

Large Subunit ◽

Critical Role ◽

Small Subunit ◽

Splicing Factor ◽

Interaction Domain ◽

Recessive Lethal ◽

Acid Fragment

ABSTRACT The heterodimeric pre-mRNA splicing factor, U2AF (U2 snRNP auxiliary factor), plays a critical role in 3′ splice site selection. Although the U2AF subunits associate in a tight complex, biochemical experiments designed to address the requirement for both subunits in splicing have yielded conflicting results. We have taken a genetic approach to assess the requirement for the Drosophila U2AF heterodimer in vivo. We developed a novel Escherichia colicopurification assay to map the domain on the DrosophilaU2AF large subunit (dU2AF50) that interacts with theDrosophila small subunit (dU2AF38). A 28-amino-acid fragment on dU2AF50 that is both necessary and sufficient for interaction with dU2AF38 was identified. Using the copurification assay, we scanned this 28-amino-acid interaction domain for mutations that abrogate heterodimer formation. A collection of these dU2AF50 point mutants was then tested in vivo for genetic complementation of a recessive lethal dU2AF50 allele. A mutation that completely abolished interaction with dU2AF38 was incapable of complementation, whereas dU2AF50 mutations that did not effect heterodimer formation rescued the recessive lethal dU2AF50 allele. Analysis of heterodimer formation in embryo extracts derived from these interaction mutant lines revealed a perfect correlation between the efficiency of subunit association and the ability to complement the dU2AF50 recessive lethal allele. These data indicate thatDrosophila U2AF heterodimer formation is essential for viability in vivo, consistent with a requirement for both subunits in splicing in vitro.

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Structural and functional consequences of the replacement of proximal residues Cys172 and Cys192 in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii

Biochemical Journal ◽

10.1042/bj20071422 ◽

2008 ◽

Vol 411 (2) ◽

pp. 241-247 ◽

Cited By ~ 8

Author(s):

María-Jesús García-Murria ◽

Saeid Karkehabadi ◽

Julia Marín-Navarro ◽

Sriram Satagopan ◽

Inger Andersson ◽

...

Keyword(s):

Chlamydomonas Reinhardtii ◽

Higher Plants ◽

Large Subunit ◽

Dimensional Structure ◽

Wild Type ◽

Wild Type Enzyme ◽

Bisphosphate Carboxylase ◽

Specificity Factor

Proximal Cys172 and Cys192 in the large subunit of the photosynthetic enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) are evolutionarily conserved among cyanobacteria, algae and higher plants. Mutation of Cys172 has been shown to affect the redox properties of Rubisco in vitro and to delay the degradation of the enzyme in vivo under stress conditions. Here, we report the effect of the replacement of Cys172 and Cys192 by serine on the catalytic properties, thermostability and three-dimensional structure of Chlamydomonas reinhardtii Rubisco. The most striking effect of the C172S substitution was an 11% increase in the specificity factor when compared with the wild-type enzyme. The specificity factor of C192S Rubisco was not altered. The Vc (Vmax for carboxylation) was similar to that of wild-type Rubisco in the case of the C172S enzyme, but approx. 30% lower for the C192S Rubisco. In contrast, the Km for CO2 and O2 was similar for C192S and wild-type enzymes, but distinctly higher (approximately double) for the C172S enzyme. C172S Rubisco showed a critical denaturation temperature approx. 2 °C lower than wild-type Rubisco and a distinctly higher denaturation rate at 55 °C, whereas C192S Rubisco was only slightly more sensitive to temperature denaturation than the wild-type enzyme. X-ray crystal structures reveal that the C172S mutation causes a shift of the main-chain backbone atoms of β-strand 1 of the α/β-barrel affecting a number of amino acid side chains. This may cause the exceptional catalytic features of C172S. In contrast, the C192S mutation does not produce similar structural perturbations.

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