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.

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 Differential N-terminal processing of beta and gamma actin in vivo

2021 ◽  
Author(s):  
Li Chen ◽  
Hsin-Yao Tang ◽  
Anna Kashina
Keyword(s):  
Amino Acid ◽  
Physiological Role ◽  
Cell Types ◽  
Amino Acid Sequences ◽  
Major Constituent ◽  
Cytoplasmic Actin ◽  
Mammalian Cell Lines ◽  
Beta Actin ◽  
Different Cell Types

AbstractActin is one of the most essential and abundant intracellular proteins, playing an essential physiological role as the major constituent of the actin cytoskeleton. Two cytoplasmic actins, beta- and gamma-actin, are encoded by different genes, but their amino acid sequences differ only by four conservative substitutions at the N-terminus, making it very difficult to dissect their individual regulation in vivo. The majority of actins are N-terminally acetylated, following the removal of N-terminal Met. Here, we analyzed beta and gamma cytoplasmic actin N-termini in vivo and found that beta actin, unlike gamma actin, specifically undergoes sequential removal of N-terminal amino acid Asp residues. This processing affects ∼1-3% of beta actin in different cell types. We identified candidate enzymes capable of mediating this type of processing, and used CRISPR/Cas-9 to delete them, individually or together, in mammalian cell lines. This deletion abolishes most of the beta actin N-terminal processing and results in changes in F-actin levels, cell spreading, filopodia formation, and cell migration, suggesting that the beta actin processing mediated by these enzymes is physiologically important to beta actin function. We propose that selective N-terminal processing of beta actin by sequential removal of Asp contributes to differentiating the functions of non-muscle actin isoforms in vivo.

Actin mRNA isoforms are differentially sorted in normal osteoblasts and sorting is altered in osteoblasts from a skeletal mutation in the rat

1998 ◽  
Vol 111 (9) ◽  
pp. 1287-1292 ◽  
Author(s):  
H. Watanabe ◽  
E.H. Kislauskis ◽  
C.A. Mackay ◽  
A. Mason-Savas ◽  
S.C. Marks
Keyword(s):  
In Situ Hybridization ◽  
Bone Resorption ◽  
Cell Types ◽  
Mrna Isoforms ◽  
Actin Isoforms ◽  
Beta Actin ◽  
Cell Processes ◽  
Actin Mrna ◽  
Actin Isoform

Actin isoform sorting has been shown to occur in a variety of cell types in culture. To this list we add osteoblasts, in which we show by in situ hybridization that beta-actin is distributed primarily in cell processes and on one side of the nucleus and gamma-actin has a perinuclear distribution. Osteoblasts from the skeletal mutation toothless (tl), evaluated under identical conditions, fail to sort these actin isoforms differentially and exhibit diffuse labeling as their major manifestation. Northern analyses of actin mRNAs showed no differences between normal and mutant cultures. Shortened osteoblast life span and an inability to direct osteoclast-mediated bone resorption have recently been demonstrated in tl mutants. The present results suggest that a failure of osteoblasts to sort actin mRNAs may be related to one or both of these pathological manifestations in this mutation and represent, to our knowledge, the first correlation of an actin mRNA-sorting abnormality with a mammalian disease.

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.

Alteration of nuclear architecture in male germ cells of chromosomally derived subfertile mice

2001 ◽  
Vol 114 (24) ◽  
pp. 4429-4434
Author(s):  
Silvia Garagna ◽  
Maurizio Zuccotti ◽  
Alan Thornhill ◽  
Raul Fernandez-Donoso ◽  
Soledad Berrios ◽  
...  
Keyword(s):  
Sertoli Cells ◽  
Spatial Organization ◽  
Nuclear Architecture ◽  
Spatial Arrangement ◽  
Cell Types ◽  
Condensed State ◽  
Germ Cell Differentiation ◽  
Y Chromosomes ◽  
Dual Colour ◽  
Pachytene Spermatocytes

The mammalian cell nucleus consists of numerous compartments involved in the regular unfolding of processes such as DNA replication and transcription, RNA maturation, protein synthesis and cell division. Knowledge is increasing of the relationships between high-order levels of chromatin organization and its spatial organization, and of how these relationships contribute to the various functions carried out in the nucleus. We have studied the spatial arrangement of mouse telocentric chromosomes 5, 11, 13, 15, 16 and 17, some of their metacentric Robertsonian derivatives, and X and Y chromosomes by whole chromosome painting in male germ (spermatogonia, pachytene spermatocytes and spermatids) and Sertoli cells of homozygous and heterozygous individuals. Using dual-colour fluorescence in situ hybridization we found that these chromosomes occupy specific nuclear territories in each cell type analysed. When chromosomes are present as Robertsonian metacentrics in the heterozygous state, that is, as Robertsonian metacentrics and their homologous telocentrics, differences in their nuclear positions are detectable: heterozygosity regularly produces a change in the nuclear position of one of the two homologous telocentrics in all the cell types studied. In the Robertsonian heterozygotes, the vast majority of the Sertoli cells show the sex chromosomes in a condensed state, whereas they appear decondensed in the Robertsonian homozygotes. As the Robertsonian heterozygosities we studied produce a chromosomally derived impairment of male germ-cell differentiation, we discuss the possibility that changes in chromosome spatial territories may alter some nuclear machinery (e.g., synapsis, differential gene expression) important for the correct unfolding of the meiotic process and for the proper functioning of Sertoli cells.

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.

Transcriptional regulation of decreased protein synthesis during skeletal muscle unloading

1989 ◽  
Vol 66 (3) ◽  
pp. 1093-1098 ◽  
Author(s):  
G. Howard ◽  
J. M. Steffen ◽  
T. E. Geoghegan
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Blot Hybridization ◽  
Qualitative Assessment ◽  
Whole Body ◽  
Dot Blot ◽  
Total Rna ◽  
Actin Mrna ◽  
Alpha Actin ◽  
Muscle Unloading

Muscle atrophy resulting from disuse is associated with marked decrements in protein synthesis. The objective of the present investigation was to determine levels of total muscle RNA and the content and composition of the mRNA fraction as a qualitative assessment of the potential regulatory role of transcriptional alterations in unloaded skeletal muscles. Hindlimb muscle unloading was produced by whole-body suspension of rats for up to 7 days. The soleus, gastrocnemius, and extensor digitorum longus (EDL) were excised from 1-, 3-, and 7-day suspended and pair-fed controls, and RNA was extracted by homogenization in 5 M guanidinium thiocyanate. Total RNA and mRNA contents were lower in soleus and gastrocnemius after 7 days of suspension compared with pair-fed controls, but total RNA and mRNA concentrations (per g muscle and per microgram total RNA, respectively) were unaltered. alpha-Actin mRNA, assessed by dot blot hybridization, was significantly reduced in soleus after 1 (37%), 3 (28%), and 7 (59%) days of suspension and in gastrocnemius after 3 (44%) and 7 (41%) days. However, alpha-actin mRNA was unchanged in the EDL after suspension. Protein synthesis directed by RNA extracted from soleus and EDL indicated marked (30–400%) alterations in mRNAs coding for several small (15- to 25-kDa) proteins. The results of this study suggest that altered transcription and availability of specific mRNAs could contribute significantly to the regulation of protein synthesis during unloading of skeletal muscle.

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.

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