Regulation of tubulin and actin mRNA production in rat brain: expression of a new beta-tubulin mRNA with development

1983 ◽  
Vol 3 (8) ◽  
pp. 1333-1342
Author(s):  
J F Bond ◽  
S R Farmer
Keyword(s):  
Rat Brain ◽  
Morphological Changes ◽  
In Vitro Translation ◽  
Brain Maturation ◽  
Cdna Clones ◽  
Mrna Levels ◽  
Beta Tubulin ◽  
Mrna Species ◽  
Tubulin Mrna ◽  
Actin Mrna

The expression of alpha-tubulin, beta-tubulin, and actin mRNA during rat brain development has been examined by using specific cDNA clones and in vitro translation techniques. During brain maturation (0 to 80 days postnatal), these mRNA species undergo a significant decrease in abundance. The kinetics of this decrease varies between the cerebrum and the cerebellum. These mRNAs are most abundant in both tissues during week 1 postnatal, each representing 10 to 15% of total mRNA activity. Both alpha- and beta-tubulin mRNA content decreases by 90 to 95% in the cerebrum after day 11 postnatal, and 70 to 80% decreases in the cerebellum after day 16. Actin sequences also decrease but to a lesser extent in both tissues (i.e., 50%). These decreases coincide with the major developmental morphological changes (i.e., neurite extension) occurring during this postnatal period. These studies have also identified the appearance of a new 2.5-kilobase beta-tubulin mRNA species, which is more predominant in the cerebellar cytoplasm. The appearance of this form occurs at a time when the major 1.8-kilobase beta-tubulin mRNA levels are declining. The possibility that the tubulin multigene family is phenotypically expressed and then this expression responds to the morphological state of the nerve cells is discussed.

scholarly journals Regulation of tubulin and actin mRNA production in rat brain: expression of a new beta-tubulin mRNA with development.

1983 ◽  
Vol 3 (8) ◽  
pp. 1333-1342 ◽  
Author(s):  
J F Bond ◽  
S R Farmer
Keyword(s):  
Rat Brain ◽  
Morphological Changes ◽  
In Vitro Translation ◽  
Brain Maturation ◽  
Cdna Clones ◽  
Mrna Levels ◽  
Beta Tubulin ◽  
Mrna Species ◽  
Tubulin Mrna ◽  
Actin Mrna

The expression of alpha-tubulin, beta-tubulin, and actin mRNA during rat brain development has been examined by using specific cDNA clones and in vitro translation techniques. During brain maturation (0 to 80 days postnatal), these mRNA species undergo a significant decrease in abundance. The kinetics of this decrease varies between the cerebrum and the cerebellum. These mRNAs are most abundant in both tissues during week 1 postnatal, each representing 10 to 15% of total mRNA activity. Both alpha- and beta-tubulin mRNA content decreases by 90 to 95% in the cerebrum after day 11 postnatal, and 70 to 80% decreases in the cerebellum after day 16. Actin sequences also decrease but to a lesser extent in both tissues (i.e., 50%). These decreases coincide with the major developmental morphological changes (i.e., neurite extension) occurring during this postnatal period. These studies have also identified the appearance of a new 2.5-kilobase beta-tubulin mRNA species, which is more predominant in the cerebellar cytoplasm. The appearance of this form occurs at a time when the major 1.8-kilobase beta-tubulin mRNA levels are declining. The possibility that the tubulin multigene family is phenotypically expressed and then this expression responds to the morphological state of the nerve cells is discussed.

scholarly journals Changes in levels of actin and tubulin mRNAs upon the lectin activation of lymphocytes.

1984 ◽  
Vol 4 (9) ◽  
pp. 1754-1760 ◽  
Author(s):  
E McCairns ◽  
D Fahey ◽  
G E Muscat ◽  
M Murray ◽  
P B Rowe
Keyword(s):  
Human Peripheral Blood ◽  
Mrna Level ◽  
Cdna Clones ◽  
Resting Cells ◽  
Beta Tubulin ◽  
Beta Actin ◽  
Rna Content ◽  
Mrna Ratio ◽  
Tubulin Mrna ◽  
Actin Mrna

The expression of beta-actin, gamma-actin, alpha-tubulin, and beta-tubulin mRNA during the lectin activation of human peripheral blood lymphocytes was examined with specific cDNA clones. The resting lymphocyte has a low level of both alpha- and beta-tubulin mRNAs, and these increase 10-fold after 72 h of lectin stimulation in which maximum cell transformation is achieved. Although there is a slight increase in tubulin mRNA during the first 6 h, most of the increase occurs between 6 and 24 h as the cells start to increase their RNA content and progress from G0 into G1. Both beta- and gamma-actin mRNAs are more abundant than the tubulin mRNAs in resting cells, with beta-actin mRNA being the major species. Upon activation, beta-actin mRNA increases threefold, whereas gamma-actin mRNA increases almost sixfold. Both beta- and gamma-actin mRNA are elevated 2.5-fold as early as 6 h, the gamma-actin mRNA level then increasing more than beta-actin between 6 and 24 h, resulting in the reduced beta-actin/gamma-actin mRNA ratio. The lectin-stimulated lymphocyte has a similar beta-actin/gamma-actin mRNA ratio as that of the human leukemic T-lymphoblast cell line CCRF-CEM. These increases are over and above the general increase in polyadenylated RNA content upon lectin activation. On returning to a noncycling state, the levels of these cytoskeletal mRNAs decrease. There were two beta-tubulin mRNAs present in lymphocyte cytoplasm, one of 1.8 kilobases and one of 2.8 kilobases in length. The nongrowing lymphocytes had relatively lower levels of the larger sized mRNA. Upon stimulation, the relative level of the larger mRNA was increased, and at 72 h the cells had approximately equal levels of both mRNAs as did the leukemic lymphoblasts.

Changes in levels of actin and tubulin mRNAs upon the lectin activation of lymphocytes

1984 ◽  
Vol 4 (9) ◽  
pp. 1754-1760
Author(s):  
E McCairns ◽  
D Fahey ◽  
G E Muscat ◽  
M Murray ◽  
P B Rowe
Keyword(s):  
Human Peripheral Blood ◽  
Mrna Level ◽  
Cdna Clones ◽  
Resting Cells ◽  
Beta Tubulin ◽  
Beta Actin ◽  
Rna Content ◽  
Mrna Ratio ◽  
Tubulin Mrna ◽  
Actin Mrna

The expression of beta-actin, gamma-actin, alpha-tubulin, and beta-tubulin mRNA during the lectin activation of human peripheral blood lymphocytes was examined with specific cDNA clones. The resting lymphocyte has a low level of both alpha- and beta-tubulin mRNAs, and these increase 10-fold after 72 h of lectin stimulation in which maximum cell transformation is achieved. Although there is a slight increase in tubulin mRNA during the first 6 h, most of the increase occurs between 6 and 24 h as the cells start to increase their RNA content and progress from G0 into G1. Both beta- and gamma-actin mRNAs are more abundant than the tubulin mRNAs in resting cells, with beta-actin mRNA being the major species. Upon activation, beta-actin mRNA increases threefold, whereas gamma-actin mRNA increases almost sixfold. Both beta- and gamma-actin mRNA are elevated 2.5-fold as early as 6 h, the gamma-actin mRNA level then increasing more than beta-actin between 6 and 24 h, resulting in the reduced beta-actin/gamma-actin mRNA ratio. The lectin-stimulated lymphocyte has a similar beta-actin/gamma-actin mRNA ratio as that of the human leukemic T-lymphoblast cell line CCRF-CEM. These increases are over and above the general increase in polyadenylated RNA content upon lectin activation. On returning to a noncycling state, the levels of these cytoskeletal mRNAs decrease. There were two beta-tubulin mRNAs present in lymphocyte cytoplasm, one of 1.8 kilobases and one of 2.8 kilobases in length. The nongrowing lymphocytes had relatively lower levels of the larger sized mRNA. Upon stimulation, the relative level of the larger mRNA was increased, and at 72 h the cells had approximately equal levels of both mRNAs as did the leukemic lymphoblasts.

scholarly journals An amino-terminal tetrapeptide specifies cotranslational degradation of beta-tubulin but not alpha-tubulin mRNAs.

1994 ◽  
Vol 14 (6) ◽  
pp. 4076-4086 ◽  
Author(s):  
C J Bachurski ◽  
N G Theodorakis ◽  
R M Coulson ◽  
D W Cleveland
Keyword(s):  
Degradation Mechanism ◽  
Full Range ◽  
Mrna Degradation ◽  
Mrna Levels ◽  
Wild Type ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Amino Terminal ◽  
Tubulin Subunit ◽  
Tubulin Mrna

The steady-state level of alpha- and beta-tubulin synthesis is autoregulated by a posttranscriptional mechanism that selectively alters alpha- and beta-tubulin mRNA levels in response to changes in the unassembled tubulin subunit concentration. For beta-tubulin mRNAs, previous efforts have shown that this is the result of a selective mRNA degradation mechanism which involves cotranslational recognition of the nascent amino-terminal beta-tubulin tetrapeptide as it emerges from the ribosome. Site-directed mutagenesis is now used to determine that the minimal sequence requirement for conferring the full range of beta-tubulin autoregulation is the amino-terminal tetrapeptide MR(E/D)I. Although tubulin-dependent changes in alpha-tubulin mRNA levels are shown to result from changes in cytoplasmic mRNA stability, transfection of wild-type and mutated alpha-tubulin genes reveals that alpha- and beta-tubulin mRNA degradation is not mediated through a common pathway. Not only does the amino-terminal alpha-tubulin tetrapeptide MREC fail to confer regulated mRNA degradation, neither wild-type alpha-tubulin transgenes nor an alpha-tubulin gene mutated to encode an amino-terminal MREI yields mRNAs that are autoregulated. Further, although slowing ribosome transit accelerates the autoregulated degradation of endogenous alpha- and beta-tubulin mRNAs, degradation of alpha-tubulin transgene mRNAs is not enhanced, and in one case, the mRNA is actually stabilized. We conclude that, despite similarities, alpha- and beta-tubulin mRNA destabilization pathways utilize divergent determinants to link RNA instability to tubulin subunit concentrations.

scholarly journals Arterial smooth muscle cells in vivo: relationship between actin isoform expression and mitogenesis and their modulation by heparin.

1988 ◽  
Vol 107 (5) ◽  
pp. 1939-1945 ◽  
Author(s):  
A W Clowes ◽  
M M Clowes ◽  
O Kocher ◽  
P Ropraz ◽  
C Chaponnier ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
In Vitro Translation ◽  
In Vivo Model ◽  
Growth Fraction ◽  
Mrna Levels ◽  
Actin Isoforms ◽  
Actin Mrna

Quiescent smooth muscle cells (SMC) in normal artery express a pattern of actin isoforms with alpha-smooth muscle (alpha SM) predominance that switches to beta predominance when the cells are proliferating. We have examined the relationship between the change in actin isoforms and entry of SMC into the growth cycle in an in vivo model of SMC proliferation (balloon injured rat carotid artery). alpha SM actin mRNA declined and cytoplasmic (beta + gamma) actin mRNAs increased in early G0/G1 (between 1 and 8 h after injury). In vivo synthesis and in vitro translation experiments demonstrated that functional alpha SM mRNA is decreased 24 h after injury and is proportional to the amount of mRNA present. At 36 h after injury, SMC prepared by enzymatic digestion were sorted into G0/G1 and S/G2 populations; only the SMC committed to proliferate (S/G2 fraction) showed a relative slight decrease in alpha SM actin and, more importantly, a large decrease in alpha SM actin mRNA. A switch from alpha SM predominance to beta predominance was present in the whole SMC population 5 d after injury. To determine if the change in actin isoforms was associated with proliferation, we inhibited SMC proliferation by approximately 80% with heparin, which has previously been shown to block SMC in late G0/G1 and to reduce the growth fraction. The switch in actin mRNAs and synthesis at 24 h was not prevented; however, alpha SM mRNA and protein were reinduced at 5 d in the heparin-treated animals compared to saline-treated controls. These results suggest that in vivo the synthesis of actin isoforms in arterial SMC depends on the mRNA levels and changes after injury in early G0/G1 whether or not the cells subsequently proliferate. The early changes in actin isoforms are not prevented by heparin, but they are eventually reversed if the SMC are kept in the resting state by the heparin treatment.

scholarly journals β-Tubulin mRNA as a Marker ofCryptosporidium parvum Oocyst Viability

1999 ◽  
Vol 65 (4) ◽  
pp. 1584-1588 ◽  
Author(s):  
Giovanni Widmer ◽  
Elizabeth A. Orbacz ◽  
Saul Tzipori
Keyword(s):  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Prolonged Incubation ◽  
Mrna Species ◽  
Viability Analysis ◽  
Viability Assay ◽  
Pcr Products ◽  
Tubulin Mrna ◽  
Β Tubulin

ABSTRACT Determining the viability of waterborne Cryptosporidium parvum oocysts remains a technical challenge. rRNA and mRNA were evaluated in a reverse transcription (RT)-PCR assay as potential markers of oocyst viability. The rationale for this approach is the rapid turnover and postmortem decay of cellular RNA. The β-tubulin mRNA and an anonymous mRNA transcript were chosen as potential markers because they are the only mRNA species in C. parvum known to possess introns. This feature facilitated the distinction between genuine RT-PCR products and PCR products originating from copurifying DNA. Prolonged incubation at room temperature of initially viable oocysts resulted in a gradual decrease in mRNA levels, which correlated with the loss of oocyst infectivity to neonatal mice. In contrast, oocysts stored at 4°C for over 39 weeks maintained their infectivity and displayed no decrease in the level of β-tubulin RT-PCR product. The postmortem decay of two mRNA species demonstrates that RT-PCR analysis can provide information on the viability of C. parvum oocysts. The methodological similarity between PCR detection and RT-PCR viability analysis could facilitate the development of a combined detection and viability assay.

Sequence heterogeneity, multiplicity, and genomic organization of alpha- and beta-tubulin genes in sea urchins

1981 ◽  
Vol 1 (12) ◽  
pp. 1125-1137
Author(s):  
D Alexandraki ◽  
J V Ruderman
Keyword(s):  
Amino Acids ◽  
Ribonucleic Acid ◽  
In Vitro Translation ◽  
Cdna Clones ◽  
Beta Tubulin ◽  
Ribonucleic Acids ◽  
Alpha Tubulin ◽  
Messenger Ribonucleic Acid ◽  
Tubulin Genes ◽  
Terminal Amino

We analyzed the multiplicity, heterogeneity, and organization of the genes encoding the alpha and beta tubulins in the sea urchin Lytechinus pictus by using cloned complementary deoxyribonucleic acid (cDNA) and genomic tubulin sequences. cDNA clones were constructed by using immature spermatogenic testis polyadenylic acid-containing ribonucleic acid as a template. alpha- and beta-tubulin clones were identified by hybrid selection and in vitro translation of the corresponding messenger ribonucleic acids, followed by immunoprecipitation and two-dimensional gel electrophoresis of the translation products. The alpha cDNA clone contains a sequence that encodes the 48 C-terminal amino acids of alpha tubulin and 104 base pairs of the 3' nontranslated portion of the messenger ribonucleic acid. The beta cDNA insertion contains the coding sequence for the 100-C terminal amino acids of beta tubulin and 83 pairs of the 3' noncoding sequence. Hybrid selections performed at different criteria demonstrated the presence of several heterogeneous, closely related tubulin messenger ribonucleic acids, suggesting the existence of heterogeneous alpha- and beta-tubulin genes. Hybridization analyses indicated that there are at least 9 to 13 sequences for each of the two tubulin gene families per haploid genome. Hybridization of the cDNA probes to both total genomic DNA and cloned germline DNA fragments gave no evidence for close physical linkage of alpha-tubulin genes with beta-tubulin genes at the DNA level. In contrast, these experiments indicated that some genes within the same family are clustered.

scholarly journals Cell-cycle-specific and serum-dependent expression of gamma-actin mRNA in Swiss mouse 3T3 cells.

1988 ◽  
Vol 8 (6) ◽  
pp. 2288-2294 ◽  
Author(s):  
A S Masibay ◽  
P K Qasba ◽  
D N Sengupta ◽  
G P Damewood ◽  
T Sreevalsan
Keyword(s):  
Growth Factor ◽  
Calcium Ionophore ◽  
Swiss Mouse ◽  
Cdna Clones ◽  
Ionophore A23187 ◽  
Resting Cells ◽  
Mrna Levels ◽  
3T3 Cells ◽  
Specific Transcript ◽  
Actin Mrna

We isolated cDNA clones that represent genes whose expression is enhanced when resting Swiss mouse 3T3 cells are stimulated to proliferate with serum. Two clones (designated pME1 and pMR6) were analyzed further. A partial sequence analysis of the pME1 insert DNA indicated that it contained a 104-base-pair stretch with extensive homology to the 3' untranslated region of gamma actin. Similar analysis of the insert DNA from the pMR6 clone indicated that it did not correspond to any previously reported gene sequence. We used the pME1 clone as a probe to determine the level of gamma actin-specific transcript in 3T3 cells under a variety of conditions. The level of gamma actin-specific mRNA began to increase in resting cells upon serum stimulation and reached a peak at 6 h. Thereafter its level declined, and by 24 h it was hardly detectable. In contrast, pMR6-specific transcript was detectable in resting cells but remained elevated even at 24 h poststimulation. The level of gamma-actin mRNA was elevated in resting cells by 12-O-tetradecanoylphorbol-13-acetate, calcium ionophore A23187, and bombesin and to a lesser extent by cholera toxin, fibroblast-derived growth factor, and dibutyryl cyclic AMP. However, insulin, vasopressin, or epidermal growth factor failed to enhance gamma-actin mRNA levels in resting cells. Inhibitors of transcription diminished the induction of gamma-actin mRNA. Gamma-actin gene was superinduced in serum-stimulated cells by cycloheximide, an inhibitor of translation. Analysis of proteins from serum-stimulated cells by two-dimensional gel electrophoresis indicated that enhanced transcription of gamma-actin mRNA resulted in a concomitant increase in the corresponding actin protein. The possible role of gamma actin, a component of the cytoskeleton, in the regulation of cell growth is discussed.

Cell-cycle-specific and serum-dependent expression of gamma-actin mRNA in Swiss mouse 3T3 cells

1988 ◽  
Vol 8 (6) ◽  
pp. 2288-2294
Author(s):  
A S Masibay ◽  
P K Qasba ◽  
D N Sengupta ◽  
G P Damewood ◽  
T Sreevalsan
Keyword(s):  
Growth Factor ◽  
Calcium Ionophore ◽  
Swiss Mouse ◽  
Cdna Clones ◽  
Ionophore A23187 ◽  
Resting Cells ◽  
Mrna Levels ◽  
3T3 Cells ◽  
Specific Transcript ◽  
Actin Mrna

We isolated cDNA clones that represent genes whose expression is enhanced when resting Swiss mouse 3T3 cells are stimulated to proliferate with serum. Two clones (designated pME1 and pMR6) were analyzed further. A partial sequence analysis of the pME1 insert DNA indicated that it contained a 104-base-pair stretch with extensive homology to the 3' untranslated region of gamma actin. Similar analysis of the insert DNA from the pMR6 clone indicated that it did not correspond to any previously reported gene sequence. We used the pME1 clone as a probe to determine the level of gamma actin-specific transcript in 3T3 cells under a variety of conditions. The level of gamma actin-specific mRNA began to increase in resting cells upon serum stimulation and reached a peak at 6 h. Thereafter its level declined, and by 24 h it was hardly detectable. In contrast, pMR6-specific transcript was detectable in resting cells but remained elevated even at 24 h poststimulation. The level of gamma-actin mRNA was elevated in resting cells by 12-O-tetradecanoylphorbol-13-acetate, calcium ionophore A23187, and bombesin and to a lesser extent by cholera toxin, fibroblast-derived growth factor, and dibutyryl cyclic AMP. However, insulin, vasopressin, or epidermal growth factor failed to enhance gamma-actin mRNA levels in resting cells. Inhibitors of transcription diminished the induction of gamma-actin mRNA. Gamma-actin gene was superinduced in serum-stimulated cells by cycloheximide, an inhibitor of translation. Analysis of proteins from serum-stimulated cells by two-dimensional gel electrophoresis indicated that enhanced transcription of gamma-actin mRNA resulted in a concomitant increase in the corresponding actin protein. The possible role of gamma actin, a component of the cytoskeleton, in the regulation of cell growth is discussed.

scholarly journals Developmental regulation of mRNA species for types II, IX and XI collagens during mouse embryogenesis

1997 ◽  
Vol 324 (1) ◽  
pp. 209-216 ◽  
Author(s):  
Merja PERÄLÄ ◽  
Mikko SAVONTAUS ◽  
Marjo METSÄRANTA ◽  
Eero VUORIO
Keyword(s):  
Developmental Regulation ◽  
Northern Analysis ◽  
Cdna Clones ◽  
Newborn Mouse ◽  
Mrna Levels ◽  
Mouse Development ◽  
Rnase Protection ◽  
Newborn Mice ◽  
Mrna Species ◽  
Collagen Mrna

Several techniques were used to study the co-ordination of mRNA levels for five constituent chains of cartilage collagen fibrils during mouse development. Short cDNA clones were first constructed for mouse and human α3(IX) and for mouse proα1(XI) collagen mRNA species. Northern analysis of developing mouse embryos revealed that the mRNA species for α1, α2 and α3 chains of type IX collagen peaked earlier than those for proα1(II) and proα1(XI) collagen chains. Quantification of these mRNA species by slot-blot hybridization confirmed this developmental regulation: the mRNA ratios for type II/type IX/type XI collagens changed from 5.7:1:0.6 (at embryonic day 12.5) to 10.6:1:0.9 (in newborn mice). However, the genes coding for the three chains of type IX collagen seemed to be under more co-ordinated regulation during mouse development. In addition to high mRNA levels in cartilages and the eye, low levels of type IX collagen transcripts were identified in brain and skin of newborn mouse using RNase protection and reverse transcriptase–PCR assays. Finally, hybridization in situ revealed identical tissue distributions of the three type IX collagen mRNA species during early chondrogenesis but somewhat more widespread expression of the α1(IX) and α3(IX) mRNA species during endochondral ossification at day 16.5 of embryonic development. These results suggest a relatively tight co-ordination of the α1(IX), α2(IX), and α3(IX) collagen mRNA species in chondrocytes, but a lack of co-ordination in several non-cartilaginous tissues.

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