Isolation and characterization of full-length cDNA clones for human alpha-, beta-, and gamma-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed

1983 ◽  
Vol 3 (5) ◽  
pp. 787-795
Author(s):  
P Gunning ◽  
P Ponte ◽  
H Okayama ◽  
J Engel ◽  
H Blau ◽  
...  
Keyword(s):  
Cdna Library ◽  
Untranslated Region ◽  
Human Muscle ◽  
Full Length ◽  
Cdna Clones ◽  
Actin Gene ◽  
Coding Region ◽  
Base Pairs ◽  
Methionine Codon ◽  
Alpha Actin

cDNA clones encoding three classes of human actins have been isolated and characterized. The first two classes (gamma and beta, cytoplasmic actins) were obtained from a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA, and the third class (alpha, muscle actin) was obtained from a cDNA library constructed from adult human muscle mRNA. A new approach was developed to enrich for full-length cDNAs. The human fibroblast cDNA plasmid library was linearized with restriction enzymes that did not cut the inserts of interest; it was then size-fractionated on gels, and the chimeric molecules of optimal length were selected for retransformation of bacteria. When the resulting clones were screened for actin-coding sequences it was found that some full-length cDNAs were enriched as much as 50- to 100-fold relative to the original frequency of full-length clones in the total library. Two types of clones were distinguished. One of these clones encodes gamma actin and contains 100 base pairs of 5' untranslated region, the entire protein coding region, and the 3' untranslated region. The second class encodes beta actin, and the longest such clone contains 45 base pairs of 5' untranslated region plus the remainder of the mRNA extending to the polyadenylic acid tail. A third class, obtained from the human muscle cDNA library, encodes alpha actin and contains 100 base pairs of 5' untranslated region, the entire coding region, and the 3' untranslated region. Analysis of the DNA sequences of the 5' end of the clones demonstrated that although beta- and gamma-actin genes start with a methionine codon (MET-Asp-Asp-Asp and MET-Glu-Glu-Glu, respectively), the alpha-actin gene starts with a methionine codon followed by a cysteine codon (MET-CYS-Asp-Glu-Asp-Glu). Since no known actin proteins start with a cysteine, it is likely that post-translational removal of cysteine in addition to methionine accompanies alpha-actin synthesis but not beta- and gamma-actin synthesis. This observation has interesting implications both for actin function and actin gene regulation and evolution.

scholarly journals Isolation and characterization of full-length cDNA clones for human alpha-, beta-, and gamma-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed.

1983 ◽  
Vol 3 (5) ◽  
pp. 787-795 ◽  
Author(s):  
P Gunning ◽  
P Ponte ◽  
H Okayama ◽  
J Engel ◽  
H Blau ◽  
...  
Keyword(s):  
Cdna Library ◽  
Untranslated Region ◽  
Human Muscle ◽  
Full Length ◽  
Cdna Clones ◽  
Actin Gene ◽  
Coding Region ◽  
Base Pairs ◽  
Methionine Codon ◽  
Alpha Actin

cDNA clones encoding three classes of human actins have been isolated and characterized. The first two classes (gamma and beta, cytoplasmic actins) were obtained from a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA, and the third class (alpha, muscle actin) was obtained from a cDNA library constructed from adult human muscle mRNA. A new approach was developed to enrich for full-length cDNAs. The human fibroblast cDNA plasmid library was linearized with restriction enzymes that did not cut the inserts of interest; it was then size-fractionated on gels, and the chimeric molecules of optimal length were selected for retransformation of bacteria. When the resulting clones were screened for actin-coding sequences it was found that some full-length cDNAs were enriched as much as 50- to 100-fold relative to the original frequency of full-length clones in the total library. Two types of clones were distinguished. One of these clones encodes gamma actin and contains 100 base pairs of 5' untranslated region, the entire protein coding region, and the 3' untranslated region. The second class encodes beta actin, and the longest such clone contains 45 base pairs of 5' untranslated region plus the remainder of the mRNA extending to the polyadenylic acid tail. A third class, obtained from the human muscle cDNA library, encodes alpha actin and contains 100 base pairs of 5' untranslated region, the entire coding region, and the 3' untranslated region. Analysis of the DNA sequences of the 5' end of the clones demonstrated that although beta- and gamma-actin genes start with a methionine codon (MET-Asp-Asp-Asp and MET-Glu-Glu-Glu, respectively), the alpha-actin gene starts with a methionine codon followed by a cysteine codon (MET-CYS-Asp-Glu-Asp-Glu). Since no known actin proteins start with a cysteine, it is likely that post-translational removal of cysteine in addition to methionine accompanies alpha-actin synthesis but not beta- and gamma-actin synthesis. This observation has interesting implications both for actin function and actin gene regulation and evolution.

scholarly journals GGeneration of an efficient artificial promoter of bovine skeletal muscle alpha-actin gene (ACTA1) through addition of cis-acting element

2015 ◽  
Vol 20 (1) ◽  
Author(s):  
Qian Hu ◽  
Huili Tong ◽  
Dandan Zhao ◽  
Yunkao Cao ◽  
Weiwei Zhang ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Core Promoter ◽  
Genetically Engineered ◽  
Actin Gene ◽  
Base Pairs ◽  
Cis Acting ◽  
The Core ◽  
Binding Factor ◽  
Bovine Skeletal Muscle ◽  
Alpha Actin

AbstractThe promoter of skeletal muscle α-actin gene (ACTA1) is highly muscle specific. The core of the bovine ACTA1 promoter extends from +29 to −233, about 262 base pairs (bp), which is sufficient to activate transcription in bovine muscle satellite cells. In this study, analysis by PCR site-specific mutagenesis showed that the cis-acting element SRE (serum response element binding factor) was processed as a transcriptional activator. In order to enhance the bovine ACTA1 promoter’s activity, we used a strategy to modify it. We cloned a fragment containing three SREs from the promoter of ACTA1, and then one or two clones were linked upstream of the core promoter (262 bp) of ACTA1. One and two clones increased the activity of the ACTA1 promoter 3-fold and 10-fold, respectively, and maintained muscle tissue specificity. The modified promoter with two clones could increase the level of ACTA1 mRNA and protein 4-fold and 1.1-fold, respectively. Immunofluorescence results showed that green fluorescence of ACTA1 increased. Additionally, the number of total muscle microfilaments increased. These genetically engineered promoters might be useful for regulating gene expression in muscle cells and improving muscle mass in livestock.

scholarly journals Evolution of the functional human beta-actin gene and its multi-pseudogene family: conservation of noncoding regions and chromosomal dispersion of pseudogenes.

1985 ◽  
Vol 5 (10) ◽  
pp. 2720-2732 ◽  
Author(s):  
S Y Ng ◽  
P Gunning ◽  
R Eddy ◽  
P Ponte ◽  
J Leavitt ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Single Copy ◽  
Functional Gene ◽  
The Other ◽  
Cdna Clones ◽  
Actin Gene ◽  
Noncoding Regions ◽  
Beta Actin ◽  
Evolutionarily Conserved ◽  
Processed Pseudogenes

We have assigned six members of the human beta-actin multigene family to specific human chromosomes. The functional gene, ACTB, is located on human chromosome 7, and the other assigned beta-actin-related sequences are dispersed over at least four different chromosomes including one locus assigned to the X chromosome. Using intervening sequence probes, we showed that the functional gene is single copy and that all of the other beta-actin related sequences are recently generated in evolution and are probably processed pseudogenes. The entire nucleotide sequence of the functional gene has been determined and is identical to cDNA clones in the coding and 5' untranslated regions. We have previously reported that the 3' untranslated region is well conserved between humans and rats (Ponte et al., Nucleic Acids Res. 12:1687-1696, 1984). Now we report that four additional noncoding regions are evolutionarily conserved, including segments of the 5' flanking region, 5' untranslated region, and, surprisingly, intervening sequences I and III. These conserved sequences, especially those found in the introns, suggest a role for internal sequences in the regulation of beta-actin gene expression.

Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen

1987 ◽  
Vol 7 (9) ◽  
pp. 3221-3230
Author(s):  
N Beauchemin ◽  
S Benchimol ◽  
D Cournoyer ◽  
A Fuks ◽  
C P Stanners
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Carcinoembryonic Antigen ◽  
Full Length ◽  
Cdna Clones ◽  
Base Pairs ◽  
Amino Terminal ◽  
Isolation And Characterization

Carcinoembryonic antigen (CEA) expression is perhaps the most prevalent of phenotypic changes observed in human cancer cells. The molecular genetic basis of this phenomenon, however, is completely unknown. Twenty-seven CEA cDNA clones were isolated from a human colon adenocarcinoma cell line. Most of these clones are full length and consist of a number (usually three) of surprisingly similar long (534 base pairs) repeats between a 5' end of 520 base pairs and a 3' end with three different termination points. The predicted translation product of these clones consists of a processed signal sequence of 34 amino acids, an amino-terminal sequence of 107 amino acids, which includes the known terminal amino acid sequence of CEA, three repeated domains of 178 amino acids each, and a membrane-anchoring domain of 27 amino acids, giving a total of 702 amino acids and a molecular weight of 72,813 for the mature protein. The repeated domains have conserved features, including the first 67 amino acids at their N termini and the presence of four cysteine residues. Comparisons with the amino acid sequences of other proteins reveals homology of the repeats with various members of the immunoglobulin supergene family, particularly the human T-cell receptor gamma chain. CEA cDNA clones in the SP-65 vector were shown to produce transcripts in vitro which could be translated in vitro to yield a protein of molecular weight 73,000 which in turn could be precipitated with CEA-specific antibodies. CEA cDNA clones were also inserted into an animal cell expression vector and introduced by transfection into mammalian cell lines. These transfectants produced a CEA-immunoprecipitable glycoprotein which could be visualized by immunofluorescence on the cell surface.

scholarly journals Systematic Assembly of a Full-Length Infectious cDNA of Mouse Hepatitis Virus Strain A59

2002 ◽  
Vol 76 (21) ◽  
pp. 11065-11078 ◽  
Author(s):  
Boyd Yount ◽  
Mark R. Denison ◽  
Susan R. Weiss ◽  
Ralph S. Baric
Keyword(s):  
Virus Strain ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Full Length ◽  
Plaque Morphology ◽  
Cdna Clones ◽  
Replicase Gene ◽  
Base Pairs ◽  
Restriction Sites ◽  
Cdna Construct

ABSTRACT A novel method was developed to assemble a full-length infectious cDNA of the group II coronavirus mouse hepatitis virus strain A59 (MHV-A59). Seven contiguous cDNA clones that spanned the 31.5-kb MHV genome were isolated. The ends of the cDNAs were engineered with unique junctions and assembled with only the adjacent cDNA subclones, resulting in an intact MHV-A59 cDNA construct of ∼31.5 kb in length. The interconnecting restriction site junctions that are located at the ends of each cDNA are systematically removed during the assembly of the complete full-length cDNA product, allowing reassembly without the introduction of nucleotide changes. RNA transcripts derived from the full-length MHV-A59 construct were infectious, although transfection frequencies were enhanced 10- to 15-fold in the presence of transcripts encoding the nucleocapsid protein N. Plaque-purified virus derived from the infectious construct replicated efficiently and displayed similar growth kinetics, plaque morphology, and cytopathology in murine cells as did wild-type MHV-A59. Molecularly cloned viruses recognized the MHV receptor (MHVR) for docking and entry, and pretreatment of cells with monoclonal antibodies against MHVR blocked virus entry and replication. Cells infected with molecularly cloned MHV-A59 virus expressed replicase (gene 1) proteins identical to those of laboratory MHV-A59. Importantly, the molecularly cloned viruses contained three marker mutations that had been derived from the engineered component clones. Full-length infectious constructs of MHV-A59 will permit genetic modifications of the entire coronavirus genome, particularly in the replicase gene. The method has the potential to be used to construct viral, microbial, or eukaryotic genomes approaching several million base pairs in length and used to insert restriction sites at any given nucleotide in a microbial genome.

scholarly journals Molecular cloning of two cysteine proteinases from paw-paw (Carica papaya)

1986 ◽  
Vol 237 (1) ◽  
pp. 105-110 ◽  
Author(s):  
R A McKee ◽  
S Adams ◽  
J A Matthews ◽  
C J Smith ◽  
H Smith
Keyword(s):  
Northern Blot ◽  
Untranslated Region ◽  
Carica Papaya ◽  
Cysteine Proteinase ◽  
Leaf Tissue ◽  
The Other ◽  
Cdna Clones ◽  
Cysteine Proteinases ◽  
Base Pairs ◽  
Hybridization Probe

Two cDNA clones for plant cysteine proteinases have been isolated from a Carica papaya (paw-paw, papaya) leaf tissue cDNA library by using a mixture of 16 synthetic oligodeoxyribonucleotides as a hybridization probe. The inserted regions are 311 and 440 base-pairs in length and have the potential to encode a region corresponding to the C-terminal region of two proteins which are homologous with the known plant cysteine proteinases and the mammalian thiol cathepsins. One of the sequences shows a high (greater than 77%) homology with the plant cysteine proteinase papain, the other is closely related to papaya chymopapain. One sequence contains all, and the other most, of the 3′ untranslated region of the mRNA. The inserts were used as specific probes in Northern Blot analyses giving an estimated size for the two mRNA species of 1.45 kilobases.

scholarly journals Functional Identification of Salt-Stress-Related Genes Using the FOX Hunting System from Ipomoea pes-caprae

2018 ◽  
Vol 19 (11) ◽  
pp. 3446 ◽  
Author(s):  
Mei Zhang ◽  
Hui Zhang ◽  
Jie-Xuan Zheng ◽  
Hui Mo ◽  
Kuai-Fei Xia ◽  
...  
Keyword(s):  
Salt Stress ◽  
Salt Tolerance ◽  
Cdna Library ◽  
Molecular Mechanisms ◽  
Full Length ◽  
Cdna Clones ◽  
Functional Screening ◽  
Yeast Mutant ◽  
Full Length Cdna ◽  
Stress Related Genes

Ipomoea pes-caprae is a seashore halophytic plant and is therefore a good model for studying the molecular mechanisms underlying salt and stress tolerance in plant research. Here, we performed Full-length cDNA Over-eXpressor (FOX) gene hunting with a functional screening of a cDNA library using a salt-sensitive yeast mutant strain to isolate the salt-stress-related genes of I. pes-caprae (IpSR genes). The library was screened for genes that complemented the salt defect of yeast mutant AXT3 and could grow in the presence of 75 mM NaCl. We obtained 38 candidate salt-stress-related full-length cDNA clones from the I. pes-caprae cDNA library. The genes are predicted to encode proteins involved in water deficit, reactive oxygen species (ROS) scavenging, cellular vesicle trafficking, metabolic enzymes, and signal transduction factors. When combined with the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses, several potential functional salt-tolerance-related genes were emphasized. This approach provides a rapid assay system for the large-scale screening of I. pes-caprae genes involved in the salt stress response and supports the identification of genes responsible for the molecular mechanisms of salt tolerance.

scholarly journals Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen.

1987 ◽  
Vol 7 (9) ◽  
pp. 3221-3230 ◽  
Author(s):  
N Beauchemin ◽  
S Benchimol ◽  
D Cournoyer ◽  
A Fuks ◽  
C P Stanners
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Carcinoembryonic Antigen ◽  
Full Length ◽  
Cdna Clones ◽  
Base Pairs ◽  
Amino Terminal ◽  
Isolation And Characterization

Carcinoembryonic antigen (CEA) expression is perhaps the most prevalent of phenotypic changes observed in human cancer cells. The molecular genetic basis of this phenomenon, however, is completely unknown. Twenty-seven CEA cDNA clones were isolated from a human colon adenocarcinoma cell line. Most of these clones are full length and consist of a number (usually three) of surprisingly similar long (534 base pairs) repeats between a 5' end of 520 base pairs and a 3' end with three different termination points. The predicted translation product of these clones consists of a processed signal sequence of 34 amino acids, an amino-terminal sequence of 107 amino acids, which includes the known terminal amino acid sequence of CEA, three repeated domains of 178 amino acids each, and a membrane-anchoring domain of 27 amino acids, giving a total of 702 amino acids and a molecular weight of 72,813 for the mature protein. The repeated domains have conserved features, including the first 67 amino acids at their N termini and the presence of four cysteine residues. Comparisons with the amino acid sequences of other proteins reveals homology of the repeats with various members of the immunoglobulin supergene family, particularly the human T-cell receptor gamma chain. CEA cDNA clones in the SP-65 vector were shown to produce transcripts in vitro which could be translated in vitro to yield a protein of molecular weight 73,000 which in turn could be precipitated with CEA-specific antibodies. CEA cDNA clones were also inserted into an animal cell expression vector and introduced by transfection into mammalian cell lines. These transfectants produced a CEA-immunoprecipitable glycoprotein which could be visualized by immunofluorescence on the cell surface.

scholarly journals The complete sequence of the mouse skeletal alpha-actin gene reveals several conserved and inverted repeat sequences outside of the protein-coding region.

1986 ◽  
Vol 6 (1) ◽  
pp. 15-25 ◽  
Author(s):  
M C Hu ◽  
S B Sharp ◽  
N Davidson
Keyword(s):  
Amino Acids ◽  
Nucleotide Sequence ◽  
Inverted Repeat ◽  
Actin Gene ◽  
Coding Region ◽  
Specific Expression ◽  
Protein Coding ◽  
Repeat Sequences ◽  
Actin Genes ◽  
Alpha Actin

The complete nucleotide sequence of a genomic clone encoding the mouse skeletal alpha-actin gene has been determined. This single-copy gene codes for a protein identical in primary sequence to the rabbit skeletal alpha-actin. It has a large intron in the 5'-untranslated region 12 nucleotides upstream from the initiator ATG and five small introns in the coding region at codons specifying amino acids 41/42, 150, 204, 267, and 327/328. These intron positions are identical to those for the corresponding genes of chickens and rats. Similar to other skeletal alpha-actin genes, the nucleotide sequence codes for two amino acids, Met-Cys, preceding the known N-terminal Asp of the mature protein. Comparison of the nucleotide sequences of rat, mouse, chicken, and human skeletal muscle alpha-actin genes reveals conserved sequences (some not previously noted) outside of the protein-coding region. Furthermore, several inverted repeat sequences, partially within these conserved regions, have been identified. These sequences are not present in the vertebrate cytoskeletal beta-actin genes. The strong conservation of the inverted repeat sequences suggests that they may have a role in the tissue-specific expression of skeletal alpha-actin genes.

scholarly journals Genome Sequence, Full-Length Infectious cDNA Clone, and Mapping of Viral Double-Stranded RNA Accumulation Determinant of Hypovirus CHV1-EP721

2006 ◽  
Vol 81 (4) ◽  
pp. 1813-1820 ◽  
Author(s):  
Haiyan Lin ◽  
Xiuwan Lan ◽  
Hong Liao ◽  
Todd B. Parsley ◽  
Donald L. Nuss ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Cdna Clone ◽  
Amino Acid Level ◽  
Cryphonectria Parasitica ◽  
Infectious Cdna Clone ◽  
Full Length ◽  
Cdna Clones ◽  
Coding Region ◽  
Viral Dsrna ◽  
Double Stranded Rna

ABSTRACT Cryphonectria parasitica strain EP721 is infected with a strain of hypovirus CHV1, CHV1-EP721, and exhibits typical hypovirulence-associated traits such as reduced pigmentation and reduced asexual sporulation. However, the accumulation of the viral double-stranded RNA (dsRNA) in this hypovirus-infected C. parasitica strain is atypically low. We now report the complete nucleotide sequence and construction of a full-length infectious cDNA clone for hypovirus CHV1-EP721. The genome sequence of CHV1-EP721 was determined to be 12,724 bp in length and to share extensive homology with two other hypovirus strains, CHV1-Euro7 and CHV1-EP713, with an average of 99% and 90% identities at the nucleotide level and 99% and 92% identities at the amino acid level, respectively. CHV1-EP721 was successfully introduced into virus-free fungal host strain EP721(-v) by transfection with transcripts derived from a full-length viral cDNA. The transfected strain had a phenotype indistinguishable from that of EP721, and the accumulation of CHV1-EP721 dsRNA in the transfectant was lower than those transfected by CHV1-Euro7 and CHV1-EP713 transcripts. Through the construction of chimeric viruses by domain swapping using infectious cDNA clones of CHV1-EP721, CHV1-EP713, and CHV1-Euro7 hypoviruses, the determinant for the low level of viral dsRNA accumulation in CHV1-EP721 was mapped to the second of two CHV1-EP721 open reading frames (ORFs), ORF B. Further refined swapping of domains within ORF B identified a 2.5-kb coding region between p48 and the polymerase domain of CHV1-EP721 as being responsible for the low viral dsRNA accumulation. Evidence is also provided that low rates of hypovirus transmission through conidial spores correlates with low viral dsRNA accumulation.

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