scholarly journals Expression cloning of oncogenes by retroviral transfer of cDNA libraries.

1995 ◽  
Vol 15 (2) ◽  
pp. 704-710 ◽  
Author(s):  
I Whitehead ◽  
H Kirk ◽  
R Kay
Keyword(s):  
Mammalian Cells ◽  
Retroviral Vectors ◽  
Cdna Libraries ◽  
Expression Cloning ◽  
Cdna Clones ◽  
Beta Catenin ◽  
Functional Screening ◽  
Nucleotide Exchange ◽  
Inhibition Of Proliferation ◽  
Retroviral Transfer

a cDNA library transfer system based on retroviral vectors has been developed for expression cloning in mammalian cells. The use of retroviral vectors results in stable cDNA transfer efficiencies which are at least 100-fold higher than those achieved by transfection and therefore enables the transfer and functional screening of very large libraries. In our initial application of retroviral transfer of cDNA libraries, we have selected for cDNAs which induce oncogenic transformation of NIH 3T3 fibroblasts, as measured by loss of contact inhibition of proliferation. Nineteen different transforming cDNAs were isolated from a total of 300,000 transferred cDNA clones. Three of these cDNAs were derived from known oncogenes (raf-1, lck, and ect2), while nine others were derived from genes which had been cloned previously but were not known to have the ability to transform fibroblasts (beta-catenin, thrombin receptor, phospholipase C-gamma 2 and Spi-2 protease inhibitor genes). The Spi-2 cDNA was expressed in antisense orientation and therefore is likely to act as an inhibitor of an endogenous transformation suppressor. Seven novel cDNAs with transforming activities, including those for three new members of the CDC24 family of guanine nucleotide exchange factors, were also cloned from the retroviral cDNA libraries. Retroviral transfer of libraries should be generally useful for cloning cDNAs which confer selectable phenotypes on many different types of mammalian cells.

scholarly journals Identification of peroxisomal proteins by using M13 phage protein VI phage display: molecular evidence that mammalian peroxisomes contain a 2,4-dienoyl-CoA reductase

1999 ◽  
Vol 340 (2) ◽  
pp. 561-568 ◽  
Author(s):  
Marc FRANSEN ◽  
Paul P. VAN VELDHOVEN ◽  
Suresh SUBRAMANI
Keyword(s):  
Mammalian Cells ◽  
Unsaturated Fatty Acids ◽  
Urate Oxidase ◽  
Cdna Libraries ◽  
Molecular Evidence ◽  
Cdna Clones ◽  
Novel Proteins ◽  
M13 Phage ◽  
Coa Reductase ◽  
Protein Vi

To elucidate unknown mammalian peroxisomal enzymes and functions, we subjected M13 phage expressing fusions between the gene encoding protein VI and a rat liver cDNA library to an immunoaffinity selection process in vitro (biopanning) with the use of antibodies raised against peroxisomal subfractions. In an initial series of biopanning experiments, four different cDNA clones were obtained. These cDNA species encoded two previously identified peroxisomal enzymes, catalase and urate oxidase, and two novel proteins that contained a C-terminal peroxisomal targeting signal (PTS1). A primary structure analysis of these novel proteins revealed that one, ending in the tripeptide AKL, is homologous to the yeast peroxisomal 2,4-dienoyl-CoA reductase (EC 1.3.1.34; DCR), an enzyme required for the degradation of unsaturated fatty acids, and that the other, ending in the tripeptide SRL, is a putative member of the short-chain dehydrogenase/reductase (SDR) family, with three isoforms. Green fluorescent protein (GFP) fusions encoding GFP-DCR-AKL, GFP-DCR, GFP-SDR-SRL and GFP-SDR were expressed in mammalian cells. The analysis of the subcellular location of the recombinant fusion proteins confirmed the peroxisomal localization of GFP-DCR-AKL and GFP-SDR-SRL, as well as the functionality of the PTS1. That the AKL protein is indeed an NADPH-dependent DCR was demonstrated by showing DCR activity of the bacterially expressed protein. These results demonstrate at the molecular level that mammalian peroxisomes do indeed contain a DCR. In addition, the results presented here indicate that the protein VI display system is suitable for the isolation of rare cDNA clones from cDNA libraries and that this technology facilitates the identification of novel peroxisomal proteins.

scholarly journals Cloning and sequence analysis of cDNAs encoding the α1, α2 and α3 chains of mouse collagen VI

1993 ◽  
Vol 291 (3) ◽  
pp. 787-792 ◽  
Author(s):  
R Z Zhang ◽  
T C Pan ◽  
R Timpl ◽  
M L Chu
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Untranslated Regions ◽  
Cdna Libraries ◽  
Cdna Clones ◽  
Collagen Vi ◽  
Central Segment ◽  
Glycosylation Sites ◽  
Key Features ◽  
Triple Helical Domain

cDNA clones encoding the alpha 1, alpha 2 and alpha 3 chains of mouse collagen VI have been isolated by screening cDNA libraries with the corresponding human probes. The composite cDNAs for the alpha 1, alpha 2, and alpha 3 chains are 2.5, 1.6 and 2.9 kb in size respectively. The alpha 1 and alpha 2 cDNAs encode the C-terminal portions of the chains as well as the entire 3′-untranslated regions, while the alpha 3 cDNAs encode a central segment of 959 amino acids flanking the triple-helical domain. The deduced amino acid sequences share 86-88% identity with the human counterparts and 67-73% identity with the chicken equivalents. Alignment of the deduced amino acid sequences of mouse, human and chicken collagens reveal that the key features of the protein, including the cysteine residues, imperfections in the Gly-Xaa-Xaa regions, Arg-Gly-Asp sequences and potential N-glycosylation sites, are mostly conserved.

scholarly journals A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells.

1983 ◽  
Vol 3 (2) ◽  
pp. 280-289 ◽  
Author(s):  
H Okayama ◽  
P Berg
Keyword(s):  
Cdna Cloning ◽  
Mammalian Cells ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Plasmid Vector ◽  
Simian Virus ◽  
Cdna Clones ◽  
Alpha Globin ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
Cloned Cdna

This paper describes a plasmid vector for cloning cDNAs in Escherichia coli; the same vector also promotes expression of the cDNA segment in mammalian cells. Simian virus 40 (SV40)-derived DNA segments are arrayed in the pcD vector to permit transcription, splicing, and polyadenylation of the cloned cDNA segment. A DNA fragment containing both the SV40 early region promoter and two introns normally used to splice the virus 16S and 19S late mRNAs is placed upstream of the cDNA cloning site to ensure transcription and splicing of the cDNA transcripts. An SV40 late region polyadenylation sequence occurs downstream of the cDNA cloning site, so that the cDNA transcript acquires a polyadenylated 3' end. By using pcD-alpha-globin cDNA as a model, we confirmed that the alpha-globin transcript produced in transfected cells is initiated correctly, spliced at either of the two introns, and polyadenylated either at the site coded in the cDNA segment or at the distal SV40 polyadenylation signal. A cDNA clone library constructed with mRNA from SV40-transformed human fibroblasts and this vector (about 1.4 X 10(6) clones) yielded full-length cDNA clones that express hypoxanthine-guanine phosphoribosyltransferase (Jolly et al., Proc. Natl. Acad. Sci. U.S.A., in press).

Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein

1992 ◽  
Vol 12 (1) ◽  
pp. 164-171
Author(s):  
M J Matunis ◽  
W M Michael ◽  
G Dreyfuss
Keyword(s):  
Hela Cells ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Rna Binding ◽  
Consensus Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Hnrnp Proteins

At least 20 major proteins make up the ribonucleoprotein (RNP) complexes of heterogeneous nuclear RNA (hnRNA) in mammalian cells. Many of these proteins have distinct RNA-binding specificities. The abundant, acidic heterogeneous nuclear RNP (hnRNP) K and J proteins (66 and 64 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are unique among the hnRNP proteins in their binding preference: they bind tenaciously to poly(C), and they are the major oligo(C)- and poly(C)-binding proteins in human HeLa cells. We purified K and J from HeLa cells by affinity chromatography and produced monoclonal antibodies to them. K and J are immunologically related and conserved among various vertebrates. Immunofluorescence microscopy with antibodies shows that K and J are located in the nucleoplasm. cDNA clones for K were isolated, and their sequences were determined. The predicted amino acid sequence of K does not contain an RNP consensus sequence found in many characterized hnRNP proteins and shows no extensive homology to sequences of any known proteins. The K protein contains two internal repeats not found in other known proteins, as well as GlyArgGlyGly and GlyArgGlyGlyPhe sequences, which occur frequently in many RNA-binding proteins. Overall, K represents a novel type of hnRNA-binding protein. It is likely that K and J play a role in the nuclear metabolism of hnRNAs, particularly for pre-mRNAs that contain cytidine-rich sequences.

An RNA-binding protein from Xenopus oocytes is associated with specific message sequences

Development ◽  
1987 ◽  
Vol 101 (4) ◽  
pp. 741-749 ◽  
Author(s):  
D.R. Crawford ◽  
J.D. Richter
Keyword(s):  
Xenopus Oocytes ◽  
Sucrose Gradient ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Cdna Libraries ◽  
Cdna Clones ◽  
Stage Of Development ◽  
Messenger Ribonucleoprotein ◽  
Stage 1

Monoclonal antibodies directed against an RNA-binding protein from Xenopus oocytes were used to immunoselect messenger ribonucleoprotein (mRNP) particles. RNA was extracted from both the immunoselected and nonselected fractions and was used to direct the synthesis of oligo (dT)-primed 32P-cDNA. These two cDNA preparations were then used to probe Xenopus stage-1 oocyte cDNA libraries to identify sequences that had been specifically coimmunoselected by the antibodies. Three cDNA clones were shown to be derived specifically from the antibody-selected mRNPs. During very early oogenesis (stage 1–2), the RNA-binding protein and the three coselected mRNAs sediment in the nontranslating mRNP region of a sucrose gradient. By oocyte stage 6, the binding protein concentration decreases by as much as 22-fold relative to polyadenylated RNA. At this stage of development, the three mRNAs are found predominantly in the polysome region of a sucrose gradient. These data demonstrate that Xenopus oocytes contain an RNA-binding protein which binds specific message sequences and may regulate their expression.

Molecular cloning and expression of rat antisecretory factor and its intracellular localization

1999 ◽  
Vol 77 (3) ◽  
pp. 223-228 ◽  
Author(s):  
Kayoko Tateishi ◽  
Yoshio Misumi ◽  
Yukio Ikehara ◽  
Kyoko Miyasaka ◽  
Akihiro Funakoshi
Keyword(s):  
Pituitary Gland ◽  
Intestinal Mucosa ◽  
Intracellular Localization ◽  
Cloning And Expression ◽  
Cdna Libraries ◽  
Cdna Clones ◽  
Cytoplasmic Protein ◽  
Rat Pituitary ◽  
26S Protease ◽  
Antisecretory Factor

Antisecretory factor (AF) was identified as a pituitary protein that inhibits the intestinal fluid secretion induced by cholera toxin. One aim of this study was to elucidate whether AF is also synthesized in the intestine or if AF produced in the pituitary is transported to the intestinal tract for its function there. cDNA clones encoding a protein proposed to be AF were isolated from rat pituitary gland and intestinal mucosa cDNA libraries. The nucleotide sequences of clones isolated from the rat pituitary gland and intestinal mucosa were identical. The deduced amino acid sequence was highly homologous to the sequence for subunit 5a of the human 26S protease that exists abundantly in the cytosol and nucleus. The production of AF in the intestine was confirmed by Northern blot and immunoblot analyses. Immunocytochemical observations of cells transfected with the rat AF cDNA showed that the AF protein was localized in the cytoplasm. These findings suggest that the protein proposed to be AF may be a cytoplasmic protein, it exists in the intestine rather than being transported from the pituitary gland, and it may function in intestinal cells.Key words: rat antisecretory factor, 26S protease, S5a, cytoplasmic protein.

scholarly journals Selective activation of a discrete family of endogenous proviral elements in normal BALB/c lymphocytes

1992 ◽  
Vol 12 (1) ◽  
pp. 220-228
Author(s):  
J A Mietz ◽  
J W Fewell ◽  
E L Kuff
Keyword(s):  
Normal Mouse ◽  
Methylation Status ◽  
Regulatory Region ◽  
Cell Types ◽  
Cdna Libraries ◽  
Cdna Clones ◽  
Sequence Specificity ◽  
Selective Activation ◽  
Mouse Tissues ◽  
R Region

Intracisternal A-particle (IAP) proviral elements are abundant and widely dispersed in the mouse genome. IAP-related transcripts have been detected in normal mouse tissues where expression is under genetic control. In this study, we sought to determine whether IAP expression in BALB/c thymus and lipopolysaccharide-stimulated B cells was due to selective or indiscriminate activation of IAP elements. cDNA libraries were prepared from each source. A total of 86 IAP cDNA clones were isolated from both libraries, and 37 of these were sequenced over a common 0.7- to 1.0-kb region of the IAP genome that included the 3' long terminal repeat (LTR). Three highly related families of elements were found to be expressed in the two cell types examined. All of the related elements had a distinctive U3 regulatory region. Thirteen individual IAP proviral elements were distinguished on the basis of sequence differences within the R region of the LTR. Hybridization of genomic DNA with element-specific oligonucleotide probes confirmed the presence of a restricted number of proviral copies in the lymphocyte-specific family of elements. Most of these copies were found to be methylated in the lymphocyte DNA, but at least seven were hypomethylated in their 5' LTRs. This study shows that activation of IAP elements in normal normal mouse lymphocytes is highly selective. Activation is probably a function of both sequence specificity and methylation status of the proviral LTR.

Further Evidence That the Majority of Primary Nuclear RNA Transcripts in Mammalian Cells Do Not Contribute to mRNA

1982 ◽  
Vol 2 (6) ◽  
pp. 701-707
Author(s):  
M. Salditt-Georgieff ◽  
J. E. Darnell
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cdna Clones ◽  
Ovary Cells ◽  
Rna Molecules ◽  
Rna Transcripts ◽  
Polyadenylic Acid ◽  
Nuclear Molecules ◽  
Nuclear Rna

Nuclear RNA from Chinese hamster ovary cells was effectively separated into polyadenylic acid [poly(A)]-containing [poly (A) + ] and non-poly(A)-containing [poly(A) − ] fractions so that ∼90% of the poly(A) was present in the (A) + fraction. Only 25% of the 5′-terminal caps of the large nuclear molecules were present in the (A) + class, but about 70% of the specific mRNA sequences (assayed with cDNA clones) were in the (A) + class. It appears that many long capped heterogeneous nuclear RNA molecules are of a different sequence category from those molecules that are successfully processed into mRNA.

Functional genomics approach to hypoxia signaling

2004 ◽  
Vol 96 (2) ◽  
pp. 765-773 ◽  
Author(s):  
Karen A. Seta ◽  
David E. Millhorn
Keyword(s):  
Cell Survival ◽  
Mammalian Cells ◽  
Cdna Libraries ◽  
Vascular Endothelial ◽  
Pheochromocytoma Cells ◽  
Adaptive Mechanisms ◽  
Endothelial Growth Factor ◽  
Microarray Analyses ◽  
Expressed Sequence ◽  
Sustained Hypoxia

Mammalian cells require a constant supply of oxygen to maintain energy balance, and sustained hypoxia can result in cell death. It is therefore not surprising that sophisticated adaptive mechanisms have evolved that enhance cell survival during hypoxia. During the past few years, there have been a growing number of reports on hypoxia-induced transcription of specific genes. In this review, we describe a unique experimental approach that utilizes focused cDNA libraries coupled to microarray analyses to identify hypoxia-responsive signal transduction pathways and genes that confer the hypoxia-tolerant phenotype. We have used the subtractive suppression hybridization (SSH) method to create a cDNA library enriched in hypoxia-regulated genes in oxygen-sensing pheochromocytoma cells and have used this library to create microarrays that allow us to examine hundreds of genes at a time. This library contains over 300 genes and expressed sequence tags upregulated by hypoxia, including tyrosine hydroxylase, vascular endothelial growth factor, and junB. Hypoxic regulation of these and other genes in the library has been confirmed by microarray, Northern blot, and real-time PCR analyses. Coupling focused SSH libraries with microarray analyses allows one to specifically study genes relevant to a phenotype of interest while reducing much of the biological noise associated with these types of studies. When used in conjunction with high-throughput, dye-based assays for cell survival and apoptosis, this approach offers a rapid method for discovering validated therapeutic targets for the treatment of cardiovascular disease, stroke, and tumors.

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