scholarly journals Nucleotide sequence and expression in vitro of cDNA derived from mRNA of int-1, a provirally activated mouse mammary oncogene.

1985 ◽  
Vol 5 (12) ◽  
pp. 3337-3344 ◽  
Author(s):  
Y K Fung ◽  
G M Shackleford ◽  
A M Brown ◽  
G S Sanders ◽  
H E Varmus
Keyword(s):  
Nucleotide Sequence ◽  
Mammary Tumor ◽  
Initiation Site ◽  
Open Reading Frame ◽  
In Vitro Translation ◽  
Cdna Clones ◽  
Coding Region ◽  
Reading Frame ◽  
Donor And Acceptor

The mouse int-1 gene is a putative mammary oncogene discovered as a target for transcriptionally activating proviral insertion mutations in mammary carcinomas induced by the mouse mammary tumor virus in C3H mice. We have isolated molecular clones of full- or nearly full-length cDNA transcribed from int-1 RNA (2.6 kilobases) in a virus-induced mammary tumor. Comparison of the nucleotide sequence of the cDNA clones with that of the int-1 gene (A. van Ooyen and R. Nusse, Cell 39:233-240, 1984) shows the following. The coding region of the int-1 gene is composed of four exons. The splice donor and acceptor sites conform to consensus; however, at least two closely spaced polyadenylation sites are used, and the transcriptional initiation site remains ambiguous. The major open reading frame is preceded by an open frame 10 codons in length. The mRNA encodes a 41-kilodalton protein with several striking features--a strongly hydrophobic amino terminus, a cysteine-rich carboxy terminus, and four potential glycosylation sites. There are no differences in nucleotide sequence between the known exons of the normal and a provirally activated allele. The length of the deduced open reading frame was further confirmed by in vitro translation of RNA transcribed from the cDNA clones with SP6 RNA polymerase.

Nucleotide sequence and expression in vitro of cDNA derived from mRNA of int-1, a provirally activated mouse mammary oncogene

1985 ◽  
Vol 5 (12) ◽  
pp. 3337-3344
Author(s):  
Y K Fung ◽  
G M Shackleford ◽  
A M Brown ◽  
G S Sanders ◽  
H E Varmus
Keyword(s):  
Nucleotide Sequence ◽  
Mammary Tumor ◽  
Initiation Site ◽  
Open Reading Frame ◽  
In Vitro Translation ◽  
Cdna Clones ◽  
Coding Region ◽  
Reading Frame ◽  
Donor And Acceptor

The mouse int-1 gene is a putative mammary oncogene discovered as a target for transcriptionally activating proviral insertion mutations in mammary carcinomas induced by the mouse mammary tumor virus in C3H mice. We have isolated molecular clones of full- or nearly full-length cDNA transcribed from int-1 RNA (2.6 kilobases) in a virus-induced mammary tumor. Comparison of the nucleotide sequence of the cDNA clones with that of the int-1 gene (A. van Ooyen and R. Nusse, Cell 39:233-240, 1984) shows the following. The coding region of the int-1 gene is composed of four exons. The splice donor and acceptor sites conform to consensus; however, at least two closely spaced polyadenylation sites are used, and the transcriptional initiation site remains ambiguous. The major open reading frame is preceded by an open frame 10 codons in length. The mRNA encodes a 41-kilodalton protein with several striking features--a strongly hydrophobic amino terminus, a cysteine-rich carboxy terminus, and four potential glycosylation sites. There are no differences in nucleotide sequence between the known exons of the normal and a provirally activated allele. The length of the deduced open reading frame was further confirmed by in vitro translation of RNA transcribed from the cDNA clones with SP6 RNA polymerase.

scholarly journals Analysis of the 3′ cis-Acting Elements of Rubella Virus by Using Replicons Expressing a Puromycin Resistance Gene

2004 ◽  
Vol 78 (5) ◽  
pp. 2553-2561 ◽  
Author(s):  
Min-Hsin Chen ◽  
Ilya Frolov ◽  
Joseph Icenogle ◽  
Teryl K. Frey
Keyword(s):  
Rubella Virus ◽  
Nonstructural Protein ◽  
Relative Survival ◽  
Open Reading Frame ◽  
In Vitro Translation ◽  
Minus Strand ◽  
Structural Protein ◽  
Coding Region ◽  
Reading Frame

ABSTRACT A rubella virus (RUB) replicon, RUBrep/PAC, was constructed and used to map the 3′ cis-acting elements (3′ CSE) of the RUB genome required for RUB replication. The RUBrep/PAC replicon had the structural protein open reading frame partially replaced by a puromycin acetyltransferase (PAC) gene. Cells transfected with RUBrep/PAC transcripts expressed the PAC gene from the subgenomic RNA, were rendered resistant to puromycin, and thus survived selection with this drug. The relative survival following puromycin selection of cells transfected with transcripts from RUBrep/PAC constructs with mutations in the 3′ CSE varied. The 3′ region necessary for optimal relative survival consisted of the 3′ 305 nucleotides (nt), a region conserved in RUB defective-interfering RNAs, and thus this region constitutes the 3′ CSE. Within the 3′ CSE, deletions in the ∼245 nt that overlap the 3′ end of the E1 gene resulted in reduced relative survivals, ranging from 20 to <1% of the parental replicon survival level while most mutations within the ∼60-nt 3′ untranslated region (UTR) were lethal. None of the 3′ CSE mutations affected in vitro translation of the nonstructural protein open reading frame (which is 5′ proximal in the genome and encodes the enzymes involved in virus RNA replication). In cells transfected with replicons with 3′ CSE mutations that survived antibiotic selection (i.e., those with mutations in the region of the 3′ CSE that overlaps the E1 coding region), the amount of replicon-specific minus-strand RNA was uniform; however, the accumulation of both plus-strand RNA species, genomic and subgenomic, varied widely, indicating that this region of the RUB 3′ CSE affects plus-strand RNA accumulation rather than minus-strand RNA synthesis.

The Drosophila suppressor of sable gene encodes a polypeptide with regions similar to those of RNA-binding proteins

1991 ◽  
Vol 11 (2) ◽  
pp. 894-905
Author(s):  
R A Voelker ◽  
W Gibson ◽  
J P Graves ◽  
J F Sterling ◽  
M T Eisenberg
Keyword(s):  
Rna Processing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Open Reading Frame ◽  
In Vitro Translation ◽  
Rna Recognition Motif ◽  
Reading Frame ◽  
Cdna Sequences ◽  
Suppressor Of Sable

The nucleotide sequence of the Drosophila melanogaster suppressor of sable [su(s)] gene has been determined. Comparison of genomic and cDNA sequences indicates that an approximately 7,860-nucleotide primary transcript is processed into an approximately 5-kb message, expressed during all stages of the life cycle, that contains an open reading frame capable of encoding a 1,322-amino-acid protein of approximately 150 kDa. The putative protein contains an RNA recognition motif-like region and a highly charged arginine-, lysine-, serine-, aspartic or glutamic acid-rich region that is similar to a region contained in several RNA-processing proteins. In vitro translation of in vitro-transcribed RNA from a complete cDNA yields a product whose size agrees with the size predicted by the open reading frame. Antisera against su(s) fusion proteins recognize the in vitro-translated protein and detect a protein of identical size in the nuclear fractions from tissue culture cells and embryos. The protein is also present in smaller amounts in cytoplasmic fractions of embryos. That the su(s) protein has regions similar in structure to RNA-processing protein is consistent with its known role in affecting the transcript levels of those alleles that it suppresses.

Pax-6, a murine paired box gene, is expressed in the developing CNS

Development ◽  
1991 ◽  
Vol 113 (4) ◽  
pp. 1435-1449 ◽  
Author(s):  
C. Walther ◽  
P. Gruss
Keyword(s):  
Amino Acids ◽  
Neural Tube ◽  
Multigene Family ◽  
Ventricular Zone ◽  
Open Reading Frame ◽  
Cdna Clones ◽  
Coding Region ◽  
Paired Domain ◽  
Reading Frame ◽  
Pax Genes

A multigene family of paired-box-containing genes (Pax genes) has been identified in the mouse. In this report, we describe the expression pattern of Pax-6 during embryogenesis and the isolation of cDNA clones spanning the entire coding region. The Pax-6 protein consists of 422 amino acids as deduced from the longest open reading frame and contains, in addition to the paired domain, a paired-type homeodomain. Beginning with day 8 of gestation, Pax-6 is expressed in discrete regions of the forebrain and the hindbrain. In the neural tube, expression is mainly confined to mitotic active cells in the ventral ventricular zone along the entire anteroposterior axis starting at day 8.5 of development. Pax-6 is also expressed in the developing eye, the pituitary and the nasal epithelium.

scholarly journals A highly conserved mouse gene with a propensity to form pseudogenes in mammals.

1988 ◽  
Vol 8 (7) ◽  
pp. 2797-2803 ◽  
Author(s):  
D L Heller ◽  
K M Gianola ◽  
L A Leinwand
Keyword(s):  
Dna Sequence ◽  
Cdna Clone ◽  
Restriction Analysis ◽  
In Vitro Transcription ◽  
Open Reading Frame ◽  
Cdna Clones ◽  
Reading Frame ◽  
Mouse Cdna ◽  
Processed Pseudogenes

A mouse cDNA clone corresponding to an abundantly transcribed poly(A)+ mRNA was found to be represented by 200 copies in mammalian genomes. To understand the origin and nature of this sequence family, we studied two genomic members and two cDNA clones from mouse liver. The DNA sequence of the coding strand of a full-length cDNA clone was shown to have an open reading frame capable of encoding a 25-kilodalton polypeptide that has not been previously described. In vitro transcription-translation experiments verified the presence of an open reading frame encoding a protein of the predicted size. Restriction analysis of genomic DNA and DNA sequence analysis of genomic clones indicated that many of the 200 members of this family represent processed pseudogenes, with one or a small number of active structural genes. The vast majority of the genomic copies are heterogeneous in length, truncated at their 5' ends with respect to the mRNA, and do not appear to have intervening sequences. Two distinct genomic members of this family were sequenced and found to represent incomplete copies of the mRNA. Both are 5' truncated at slightly different points with respect to the mRNA. Both pseudogenes have multiple base changes, insertions, and deletions relative to the mRNA, and one of them encodes the poly(A) tail of the mRNA. The expression of this gene family is highest in rapidly dividing cells such as early mouse embryos and testis, but was seen in all tissues tested. This gene shows extremely high sequence conservation, extending to chicken, amphibian, and nematode genomes. Surprisingly, the gene appears to exist in only one copy in these organisms.

A highly conserved mouse gene with a propensity to form pseudogenes in mammals

1988 ◽  
Vol 8 (7) ◽  
pp. 2797-2803
Author(s):  
D L Heller ◽  
K M Gianola ◽  
L A Leinwand
Keyword(s):  
Dna Sequence ◽  
Cdna Clone ◽  
Restriction Analysis ◽  
In Vitro Transcription ◽  
Open Reading Frame ◽  
Cdna Clones ◽  
Reading Frame ◽  
Mouse Cdna ◽  
Processed Pseudogenes

A mouse cDNA clone corresponding to an abundantly transcribed poly(A)+ mRNA was found to be represented by 200 copies in mammalian genomes. To understand the origin and nature of this sequence family, we studied two genomic members and two cDNA clones from mouse liver. The DNA sequence of the coding strand of a full-length cDNA clone was shown to have an open reading frame capable of encoding a 25-kilodalton polypeptide that has not been previously described. In vitro transcription-translation experiments verified the presence of an open reading frame encoding a protein of the predicted size. Restriction analysis of genomic DNA and DNA sequence analysis of genomic clones indicated that many of the 200 members of this family represent processed pseudogenes, with one or a small number of active structural genes. The vast majority of the genomic copies are heterogeneous in length, truncated at their 5' ends with respect to the mRNA, and do not appear to have intervening sequences. Two distinct genomic members of this family were sequenced and found to represent incomplete copies of the mRNA. Both are 5' truncated at slightly different points with respect to the mRNA. Both pseudogenes have multiple base changes, insertions, and deletions relative to the mRNA, and one of them encodes the poly(A) tail of the mRNA. The expression of this gene family is highest in rapidly dividing cells such as early mouse embryos and testis, but was seen in all tissues tested. This gene shows extremely high sequence conservation, extending to chicken, amphibian, and nematode genomes. Surprisingly, the gene appears to exist in only one copy in these organisms.

Nucleotide sequence of the cDNA encoding the common α subunit of the chicken pituitary glycoprotein hormones

1992 ◽  
Vol 8 (1) ◽  
pp. 21-27 ◽  
Author(s):  
D. N. Foster ◽  
D. Galehouse ◽  
T. Giordano ◽  
B. Min ◽  
I. C. Lamb ◽  
...  
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Cdna Clones ◽  
Glycoprotein Hormones ◽  
Coding Region ◽  
Α Subunit ◽  
Reading Frame ◽  
Pituitary Glands ◽  
Steady State Levels ◽  
Gonadotrophin Releasing Hormone

ABSTRACT Recombinant cDNA clones that encode the α subunit of the chicken pituitary glycoprotein hormones were isolated from a pituitary library. The longer of the two cDNA clones that were sequenced was 754bp in length. It contained 81 nucleotides of the 5′-untranslated region (UTR), an open-reading frame of 360bp that encoded a 24 amino acid leader polypeptide sequence as well as the 96 amino acid mature α subunit, and 268 nucleotides of the 3′-UTR, followed by a 45 bp poly(A) tract. There was 69–79% homology between the nucleotide sequence of the coding region for the chicken and mammalian α-subunit cDNAs. Northern blot analysis revealed that the steady-state levels of an approximately 800 bp α-subunit specific transcript increased quantitatively when dispersed chicken pituitary glands were treated in culture with chicken gonadotrophin-releasing hormone-I.

scholarly journals The Drosophila suppressor of sable gene encodes a polypeptide with regions similar to those of RNA-binding proteins.

1991 ◽  
Vol 11 (2) ◽  
pp. 894-905 ◽  
Author(s):  
R A Voelker ◽  
W Gibson ◽  
J P Graves ◽  
J F Sterling ◽  
M T Eisenberg
Keyword(s):  
Rna Processing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Open Reading Frame ◽  
In Vitro Translation ◽  
Rna Recognition Motif ◽  
Reading Frame ◽  
Cdna Sequences ◽  
Suppressor Of Sable

The nucleotide sequence of the Drosophila melanogaster suppressor of sable [su(s)] gene has been determined. Comparison of genomic and cDNA sequences indicates that an approximately 7,860-nucleotide primary transcript is processed into an approximately 5-kb message, expressed during all stages of the life cycle, that contains an open reading frame capable of encoding a 1,322-amino-acid protein of approximately 150 kDa. The putative protein contains an RNA recognition motif-like region and a highly charged arginine-, lysine-, serine-, aspartic or glutamic acid-rich region that is similar to a region contained in several RNA-processing proteins. In vitro translation of in vitro-transcribed RNA from a complete cDNA yields a product whose size agrees with the size predicted by the open reading frame. Antisera against su(s) fusion proteins recognize the in vitro-translated protein and detect a protein of identical size in the nuclear fractions from tissue culture cells and embryos. The protein is also present in smaller amounts in cytoplasmic fractions of embryos. That the su(s) protein has regions similar in structure to RNA-processing protein is consistent with its known role in affecting the transcript levels of those alleles that it suppresses.

scholarly journals Three Spliced mRNAs of TT Virus Transcribed from a Plasmid Containing the Entire Genome in COS1 Cells

2000 ◽  
Vol 74 (21) ◽  
pp. 9980-9986 ◽  
Author(s):  
Toshio Kamahora ◽  
Shigeo Hino ◽  
Hironori Miyata
Keyword(s):  
Consensus Sequence ◽  
Open Reading Frame ◽  
Whole Genome ◽  
Transcription Profile ◽  
Coding Region ◽  
Entire Genome ◽  
Reading Frame ◽  
Tt Virus ◽  
Donor And Acceptor ◽  
The Family

ABSTRACT A permuted whole-genome construct of a TT virus (TTV), named VT416, had 3,852 nucleotides (nt) 98.2% similar to the prototype TA278 genome. To allow the transcription of TTV from the internal promoter, pBK*VT416(1.3G), carrying 1.3 units of VT416, was constructed. The poly(A)+ RNAs expressed in COS1 cells 48 h posttransfection contained three TTV mRNA species 3.0, 1.2, and 1.0 kb in length, which were recovered in the 13 DNA clones from a λ phage cDNA library. These mRNAs in the antigenomic orientation possessed in common the 3′ terminus downstream of a poly(A) signal (A3073ATAAA) and the 5′ terminus downstream of a cap site (C98ACTTC). A common splicing to join nt 185 with nt 277 was detected in all mRNAs. The coding region of the largest open reading frame (ORF) was maintained in 3.0-kb mRNA, because this splicing was located upstream of its initiation codon (A589TG). The second splicing was detected in 1.2-kb mRNA to join nt 711 with nt 2374 and in 1.0-kb mRNA to bind nt 711 to nt 2567. They linked a proposed ORF2 to another ORF for creating new ORFs over nt 2374 to 2872 in frame 2 and nt 2567 to 3074 in frame 3. The donor and acceptor sites of all three splicings matched the consensus sequence and were conserved in most of the 16 TTVs of distinct genotypes retrieved from the database. The observed transcription profile is unique to TTV among known members in the familyCircoviridae.

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