Further Studies on the Differences in the Interaction of Simian Virus 40 with African Green Monkey Kidney and Human Diploid Cells

We have investigated the site of surface expression of the neuraminidase (NA) glycoprotein of influenza A virus, which, in contrast to the hemagglutinin, is bound to membranes by hydrophobic residues near the NH2-terminus. Madin-Darby canine kidney or primary African green monkey kidney cells infected with influenza A/WSN/33 virus and subsequently labeled with monoclonal antibody to the NA and then with a colloidal gold- or ferritin-conjugated second antibody exhibited specific labeling of apical surfaces. Using simian virus 40 late expression vectors, we also studied the surface expression of the complete NA gene (SNC) and a truncated NA gene (SN10) in either primary or a polarized continuous line (MA104) of African green monkey kidney cells. The polypeptides encoded by the cloned NA cDNAs were expressed on the surface of both cell types. Analysis of [3H]mannose-labeled polypeptides from recombinant virus-infected MA104 cells showed that the products of cloned NA cDNA comigrated with glycosylated NA from influenza virus-infected cells. Both the complete and the truncated glycoproteins were found to be preferentially expressed on apical plasma membranes, as detected by immunogold labeling. These results indicate that the NA polypeptide contains structural features capable of directing the transport of the protein to apical cell surfaces and the first 10 amino-terminal residues of the NA polypeptide are not involved in this process.

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Efficient transfer of cloned DNA into human diploid cells: protoplast fusion in suspension

Molecular and Cellular Biology ◽

10.1128/mcb.4.11.2549-2552.1984 ◽

1984 ◽

Vol 4 (11) ◽

pp. 2549-2552

Author(s):

P Litzkas ◽

K K Jha ◽

H L Ozer

Keyword(s):

Simian Virus 40 ◽

Cell Types ◽

Simian Virus ◽

Selective Medium ◽

T Antigen ◽

Human Diploid Fibroblasts ◽

Rous Sarcoma ◽

Sarcoma Virus ◽

Human Diploid Cells ◽

Diploid Cells

A method for fusion of protoplasts bearing amplified plasmids and human diploid fibroblasts or other cell types in suspension is described. Transient expression of plasmid-encoded proteins occurs in up to 50% of the human cells, as demonstrated for simian virus 40 T antigen by immunofluorescence and the Escherichia coli xanthine-guanine phosphoribosyl transferase by autoradiography. In contrast, frequencies of stable transformants were similar to those obtained by the CaPO4 coprecipitation technique. However, experiments with both methods involving the recombinant pRSVneo (in which the Rous sarcoma virus long terminal repeat regulates expression of the antibiotic-inactivating aminoglycoside phosphotransferase) revealed a much higher frequency of colonies in G418 selective medium with constructions in which the early region of simian virus 40 DNA was present as well. We propose a role for the simian virus 40 T antigen in enhancing stable transformation in this system.

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Preparation of a "functional library" of African green monkey DNA fragments which substitute for the processing/polyadenylation signal in the herpes simplex virus type 1 thymidine kinase gene

Molecular and Cellular Biology ◽

10.1128/mcb.3.4.643-653.1983 ◽

1983 ◽

Vol 3 (4) ◽

pp. 643-653

Author(s):

G M Santangelo ◽

C N Cole

Keyword(s):

Gene Expression ◽

Thymidine Kinase ◽

Low Frequency ◽

Simian Virus 40 ◽

Simian Virus ◽

African Green Monkey ◽

Dna Fragments ◽

Coding Region ◽

Kinase Gene ◽

Green Monkey

Fragments of African green monkey (Cercopithecus aethiops) DNA (3.5 to 18.0 kilobases) were inserted downstream from the thymidine kinase (TK, tk) coding region in pTK206/SV010, a gene construct which lacks both copies of the hexanucleotide 5'-AATAAA-3' and contains a simian virus 40 origin of replication, allowing it to replicate in Cos-1 cells. No polyadenylated tk mRNA was detected in Cos-1 cells transfected by pTK206/SV010. The ability of simian DNA fragments to restore tk gene expression was examined by measuring the incorporation of [125I]iododeoxycytidine into DNA in Cos-1 cells transfected by pTK206/SV010 insertion derivatives. tk gene expression was restored by the insertion in 56 of the 67 plasmids analyzed, and the level of expression equaled or exceeded that obtained with the wild-type tk gene in 30 of these. In all plasmids examined that showed restoration of tk gene expression, polyadenylated tk mRNA of discrete size was detected. The sizes of these tk mRNAs were consistent with the existence of processing and polyadenylation signals within the inserted DNA fragments. The frequency with which inserted fragments restored tk gene expression suggests that the minimal signal for processing and polyadenylation is a hexanucleotide (AAUAAA or a similar sequence). LTK- cells were biochemically transformed to TK+ with representative insertion constructs. pTK206/SV010 transformed LTK- cells at a very low frequency; the frequency of transformation with insertion derivatives was 40 to 12,000 times higher.

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Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells.

Molecular and Cellular Biology ◽

10.1128/mcb.2.9.1044 ◽

1982 ◽

Vol 2 (9) ◽

pp. 1044-1051 ◽

Cited By ~ 3916

Author(s):

C M Gorman ◽

L F Moffat ◽

B H Howard

Keyword(s):

Promoter Region ◽

Mammalian Cells ◽

Simian Virus 40 ◽

Simian Virus ◽

Chloramphenicol Acetyltransferase ◽

Repeat Sequence ◽

Monkey Kidney ◽

Sv40 Promoter ◽

Hindiii Site ◽

African Green Monkey Kidney

We constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. We also constructed a recombinant, pSV0-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.

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African Green Monkey Fibroblast Action Morphology during SV40 Infection

Zeitschrift für Naturforschung C ◽

10.1515/znc-1977-5-615 ◽

1977 ◽

Vol 32 (5-6) ◽

pp. 409-412

Author(s):

Gerhard Brandner ◽

Myung-Sam Cho

Keyword(s):

Tumor Antigen ◽

Actin Filaments ◽

Simian Virus 40 ◽

Simian Virus ◽

African Green Monkey ◽

Skin Fibroblasts ◽

Focus Formation ◽

Normal Morphology ◽

Green Monkey ◽

Sv40 Infection

Abstract Monkey skin fibroblasts were infected with simian virus 40. Cells that exhibited the viral tumor antigen were found to retain the normal morphology of actin filaments up to six days after infection. However when cells were transformed in terms of focus formation they had lost the normal actin morphology.

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