Gestational changes in Ca2+ transport across rat placenta and mRNA for calbindin9K and Ca(2+)-ATPase

1992 ◽  
Vol 263 (4) ◽  
pp. R930-R935 ◽  
Author(s):  
J. D. Glazier ◽  
D. E. Atkinson ◽  
K. L. Thornburg ◽  
P. T. Sharpe ◽  
D. Edwards ◽  
...  
Keyword(s):  
Diffusion Coefficient ◽  
Calcium Transport ◽  
Hybridization Analysis ◽  
Northern Hybridization ◽  
Gestational Period ◽  
Beta Actin ◽  
Mrna Ratio ◽  
Calcium Clearance ◽  
Actin Mrna ◽  
Rate Limiting

The unidirectional maternofetal clearance (Kmf) of 45Ca was measured across the rat placenta over the last one-third of gestation. Kmf for 45Ca normalized to its diffusion coefficient in water (Kmf/Dw) increased 72-fold between days 15 and 22 of gestation from 3.5 +/- 0.3 to 253.1 +/- 22.0 cm/g placenta, respectively. At 15 and 18 days of gestation, Kmf/Dw for 45Ca was similar to Kmf/Dw for the paracellular marker [14C]mannitol, but at 21 and 22 days of gestation, Kmf/Dw for 45Ca was significantly higher than Kmf/Dw for [14C]mannitol, indicating that an additional route of transfer, other than diffusion, becomes available to calcium during this period. Northern hybridization analysis demonstrated that rat placental calbindin9K-to-beta-actin mRNA ratio increased 135-fold between 15 and 22 days of gestation and was temporally associated with the gestational increase in Kmf/Dw for 45Ca. In contrast, rat placental Ca(2+)-ATPase-to-beta-actin mRNA ratio increased only two- to threefold over the same gestational period and did not mirror the gestational changes in calcium clearance. These trends suggest that the expression of placental calbindin9K, but not Ca(2+)-ATPase, may be rate limiting to placental calcium transport in the rat.

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.

Cold-induced alterations in uncoupling protein and its mRNA are seasonally dependent in ground squirrels

1995 ◽  
Vol 269 (2) ◽  
pp. R357-R364
Author(s):  
S. E. Nizielski ◽  
C. J. Billington ◽  
A. S. Levine
Keyword(s):  
Cold Exposure ◽  
Uncoupling Protein ◽  
Late Summer ◽  
Ground Squirrels ◽  
Mitochondrial Uncoupling ◽  
Mitochondrial Uncoupling Protein ◽  
Beta Actin ◽  
Mrna Ratio ◽  
Brown Adipose ◽  
Actin Mrna

We were interested in determining whether season affects the ability of cold exposure to increase brown adipose tissue (BAT) thermogenic function in 13-lined ground squirrels after acute and chronic cold (4 degrees C) exposure. Tissues were collected from animals in April and September after cold exposure for 12, 24, or 48 h. Animals chronically exposed to the cold (10 days) were killed in early May and mid-August. We found that mitochondrial uncoupling protein (UCP) concentrations varied seasonally, with concentrations in control animals (at 23 degrees C) higher in late summer (mid-August and September) than in the spring (April and early May). Cold exposure in late summer did not induce further increases in UCP concentrations. In contrast, when animals were cold exposed in the spring, UCP concentrations and total UCP increased. Surprisingly, 10 days at 4 degrees C did not cause a greater increase in UCP concentrations than did 24 h at 4 degrees C. Chronic cold exposure increased the UCP mRNA-to-beta-actin mRNA ratio 48% in May, whereas a fivefold increase occurred in August. GDP binding was increased after 12 h at 4 degrees C in April; in contrast, animals attempted to hibernate when placed in the cold in September, and no increase in GDP binding was observed. Chronic cold exposure caused GDP binding to increase at both times. These results indicate that mitochondrial UCP concentrations are seasonally regulated in the 13-lined ground squirrel.

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 Characterization of a beta-actin mRNA zipcode-binding protein.

1997 ◽  
Vol 17 (4) ◽  
pp. 2158-2165 ◽  
Author(s):  
A F Ross ◽  
Y Oleynikov ◽  
E H Kislauskis ◽  
K L Taneja ◽  
R H Singer
Keyword(s):  
Nuclear Export ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Affinity Purification ◽  
Nuclear Export Signal ◽  
Degenerate Primers ◽  
Band Shift ◽  
Beta Actin ◽  
Actin Mrna

Localization of beta-actin mRNA to the leading edge of fibroblasts requires the presence of conserved elements in the 3' untranslated region of the mRNA, including a 54-nucleotide element which has been termed the "zipcode" (E. Kislauskis, X. Zhu, and R. H. Singer, J. Cell Biol. 127:441-451, 1994). In order to identify proteins which bind to the zipcode and possibly play a role in localization, we performed band-shift mobility assays, UV cross-linking, and affinity purification experiments. A protein of 68 kDa was identified which binds to the proximal (to the coding region) half of the zipcode with high specificity (ZBP-1). Microsequencing provided unique peptide sequences of approximately 15 residues each. Degenerate primers corresponding to the codons derived from the peptides were synthesized and used for PCR amplification. Screening of a chicken cDNA library resulted in isolation of several clones providing a DNA sequence encoding a 67.7-kDa protein with regions homologous to several RNA-binding proteins, such as hnRNP E1 and E2, and with consensus mRNA recognition motif with RNP1 and 2 motifs and a putative REV-like nuclear export signal. Antipeptide antibodies were raised in rabbits which bound to ZBP-1 and coimmunoprecipitated proteins of 120 and 25 kDa. The 120-kDa protein was also obtained by affinity purification with the RNA zipcode sequence, along with a 53-kDa protein, but the 25-kDa protein appeared only in immunoprecipitations. Mutation of one of the conserved sequences within the zipcode, an ACACCC element in its proximal half, greatly reduced its protein binding and localization properties. These data suggest that the 68-kDa ZBP-1 we have isolated and cloned is an RNA-binding protein that functions within a complex to localize beta-actin mRNA.

An amphibian cytoskeletal-type actin gene is expressed exclusively in muscle tissue

Development ◽  
1987 ◽  
Vol 101 (2) ◽  
pp. 393-402 ◽  
Author(s):  
T.J. Mohun ◽  
N. Garrett
Keyword(s):  
Nucleotide Sequence ◽  
Protein Isoforms ◽  
Actin Gene ◽  
Promoter Regions ◽  
Conserved Sequence ◽  
Beta Actin ◽  
Genes Encoding ◽  
Actin Mrna ◽  
Ccaat Box ◽  
Equivalent Region

The complete nucleotide sequence of two Xenopus actin genes encoding cytoskeletal protein isoforms has been determined. Transcripts from these genes are remarkably similar in nucleotide sequence throughout their length and code for type-5 and type-8 cytoskeletal actins. Both share some sequence homology with human gamma-actin mRNA within the 3′ untranslated region but none with the equivalent region of any vertebrate beta-actin transcript. The promoter regions of the two Xenopus genes are virtually identical from the cap site to the CCAAT box and show extensive homology further upstream. Despite such similarity, the two genes are divergently expressed during embryonic development. The type-5 actin gene is expressed in all regions of the developing embryo whilst the type-8 gene is coregulated with the muscle-specific skeletal actin gene. In common with mammalian and avian cytoskeletal actin counterparts, the Xenopus genes possess a conserved sequence within their promoter that has previously been identified as a transcription-factor-binding site.

Molecular cloning and characterization of mutant and wild-type human beta-actin genes

1984 ◽  
Vol 4 (10) ◽  
pp. 1961-1969
Author(s):  
J Leavitt ◽  
P Gunning ◽  
P Porreca ◽  
S Y Ng ◽  
C S Lin ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Human Fibroblasts ◽  
Actin Gene ◽  
Actin Genes ◽  
Beta Actin ◽  
Gene Libraries ◽  
Actin Mrna ◽  
Dna Fragment ◽  
Specific Sequences

There are more than 20 beta-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional beta-actin genes, we used the new method of B. Seed (Nucleic Acids Res. 11:2427-2446, 1983) for selecting genomic clones by homologous recombination. A derivative of the pi VX miniplasmid, pi AN7 beta 1, was constructed by insertion of the 600-base-pair 3' untranslated region of the beta-actin mRNA expressed in human fibroblasts. Five clones containing beta-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete beta-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then used to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant beta-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived clones verified the identity of the beta-actin gene expressed in human fibroblasts.

scholarly journals Simultaneous in situ detection of two mRNAs in the same cell using riboprobes labeled with biotin and 35S.

1990 ◽  
Vol 38 (7) ◽  
pp. 917-922 ◽  
Author(s):  
S Ozden ◽  
C Aubert ◽  
D Gonzalez-Dunia ◽  
M Brahic
Keyword(s):  
In Situ Hybridization ◽  
Theiler's Virus ◽  
Bhk Cells ◽  
Beta Actin ◽  
Actin Mrna ◽  
High Level ◽  
Radiolabeled Probe ◽  
In Situ Detection

We used 35S-labeled and biotinylated cRNAs (riboprobes) to detect simultaneously two different mRNAs by in situ hybridization. In a first step we established the conditions under which each type of probe achieved the same high level of sensitivity. We then used these conditions to hybridize BHK cells infected with Theiler's virus, a murine picornavirus, with a mixture of a virus-specific biotinylated riboprobe and a 35S-labeled riboprobe specific for beta-actin mRNA. Both mRNAs could be detected in the same cell, although the sensitivity achieved by the radiolabeled probe was reduced by about 40% by the simultaneous hybridization with the biotinylated probe.

scholarly journals Mouse testes contain two size classes of actin mRNA that are differentially expressed during spermatogenesis.

1985 ◽  
Vol 5 (7) ◽  
pp. 1649-1654 ◽  
Author(s):  
S H Waters ◽  
R J Distel ◽  
N B Hecht
Keyword(s):  
Untranslated Region ◽  
Cell Types ◽  
Dot Blot ◽  
Size Classes ◽  
Cytoplasmic Actin ◽  
Beta Actin ◽  
Actin Mrna ◽  
Residual Bodies ◽  
Pachytene Spermatocytes ◽  
Sexually Mature

Using several actin isotype-specific cDNA probes, we found actin mRNA of two size classes, 2.1 and 1.5 kilobases (kb), in extracts of polyadenylated and nonpolyadenylated RNA from sexually mature CD-1 mouse testes. Although the 2.1-kb sequence was present in both meiotic and postmeiotic testicular cell types, it decreased manyfold in late haploid cells. The 1.5-kb actin sequence was not detectable in meiotic pachytene spermatocytes (or in liver or kidney cells), but was present in round and elongating spermatids and residual bodies. To differentiate between the beta- and gamma-actin mRNAs, we isolated a cDNA, pMGA, containing the 3' untranslated region of a mouse cytoplasmic actin that has homology to the 3' untranslated region of a human gamma-actin cDNA but not to the 3' untranslated regions of human alpha-, beta-, or cardiac actins. Dot blot hybridizations with pMGA detected high levels of presumptive gamma-actin mRNA in pachytene spermatocytes and round spermatids, with lower amounts found in elongating spermatids. Hybridization with the 3' untranslated region of a rat beta-actin probe revealed that round spermatids contained higher levels of beta-actin mRNA than did pachytene spermatocytes or residual bodies. Both probes hybridized to the 2.1-kb actin mRNA but failed to hybridize to the 1.5-kb mRNA.

scholarly journals Characterization of the Ustilago maydis sid2 Gene, Encoding a Multidomain Peptide Synthetase in the Ferrichrome Biosynthetic Gene Cluster

2001 ◽  
Vol 183 (13) ◽  
pp. 4040-4051 ◽  
Author(s):  
Walter M. Yuan ◽  
Guillaume D. Gentil ◽  
Allen D. Budde ◽  
Sally A. Leong
Keyword(s):  
Amino Acids ◽  
Cyclic Peptide ◽  
Ustilago Maydis ◽  
Nonribosomal Peptide Synthetase ◽  
Hybridization Analysis ◽  
Northern Hybridization ◽  
Nucleotide Sequencing ◽  
Biosynthetic Gene ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase

ABSTRACT Ustilago maydis, the causal agent of corn smut disease, acquires and transports ferric ion by producing the extracellular, cyclic peptide, hydroxamate siderophores ferrichrome and ferrichrome A. Ferrichrome biosynthesis likely proceeds by hydroxylation and acetylation of l-ornithine, and later steps likely involve covalently bound thioester intermediates on a multimodular, nonribosomal peptide synthetase. sid1 encodesl-ornithine N 5-oxygenase, which catalyzes hydroxylation of l-ornithine, the first committed step of ferrichrome and ferrichrome A biosynthesis in U. maydis. In this report we characterize sid2, another biosynthetic gene in the pathway, by gene complementation, gene replacement, DNA sequence, and Northern hybridization analysis. Nucleotide sequencing has revealed that sid2 is located 3.7 kb upstream of sid1 and encodes an intronless polypeptide of 3,947 amino acids with three iterated modules of an approximate length of 1,000 amino acids each. Multiple motifs characteristic of the nonribosomal peptide synthetase protein family were identified in each module. A corresponding iron-regulated sid2 transcript of 11 kb was detected by Northern hybridization analysis. By contrast, constitutive accumulation of this large transcript was observed in a mutant carrying a disruption of urbs1, a zinc finger, GATA family transcription factor previously shown to regulate siderophore biosynthesis in Ustilago. Multiple GATA motifs are present in the intergenic region between sid1 and sid2, suggesting bidirectional transcription regulation by urbs1of this pathway. Indeed, mutation of two of these motifs, known to be important to regulation of sid1, altered the differential regulation of sid2 by iron.

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