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Biomedical subjects

B Sugden

Publications and source records attributed to B Sugden.

87 records · Page 5Linked to original sources
PubMed1977

Clonal transformation of adult human leukocytes by Epstein-Barr virus.

We have developed a clonal transformation assay for Epstein-Barr virus which uses adult human leukocytes as target cells. The target cells were isolated from Epstein-Barr seronegative donors, and the same donor's cells could be studied repeatedly over long periods of time. When these cells were transformed by Epstein-Barr virus and had proliferated sufficiently to be studied, they had an average cloning efficiency of 3%. Assuming this average cloning efficiency obtains at the onset of transformation, we calculate that transformation by Epstein-Barr virus leads to immortalization maximally of about 1 in 30 of the adult peripheral leukocytes exposed to the virus. Studying the number of colonies transformed as a function of the amount of virus to which the cells are exposed indicates that a single DNA-containing virus particle is sufficient to transform a cell. All of the transformed clones studied harbored viral DNA. This technique will now permit, for the first time, our studying clonal variations in adult peripheral leukocytes transformed by Epstein-Barr virus as a function of input multiplicity of the virus and of the donor's immune status.

Adult↗
PubMed1976

Nucleic acid renaturation and restriction endonuclease cleavage analyses show that the DNAs of a transforming and a nontransforming strain of Epstein-Barr virus share approximately 90% of their nucleotide sequences.

Viral DNA molecules were purified from a nontransforming and a transforming strain of Epstein-Barr virus. Each viral DNA was labeled in vitro and renatured in the presence of an excess of either one or the other unlabeled viral DNA. Both viral DNAs were also digested with the Eco R1 restriction endonuclease and subsequently labeled by using avian myeloblastosis virus DNA polymerase to repair either the EcoR1 nuclease-generated single-stranded ends of the DNAs or their single-stranded ends produced by a second digestion with exonuclease III after the first EcoR1 nuclease digestion. The results of these experiments support three general conclusions: (i) the DNAs of these two strains of Epstein-Barr virus share approximately 90% of their nucleotide sequences; (ii) both viral DNA populations are reasonably homogenous; and (iii) both DNAs contain repetitions or inverted repetitions of some of their nucleotide sequences.

Base Sequence↗
PubMed1974

Viral DNA in transformed cells. III. The amounts of different regions of the SV40 genome present in a line of transformed mouse cells.

(32)P-Labeled SV40 DNA was treated sequentially with restricting endonucleases EcoRI and Hpa I, and the resulting four fragments of DNA were separated by gel electrophoresis. The kinetics of renaturation of each of the fragments and of complete SV40 DNA were measured in the presence of DNA extracted from the SVT2 line of SV40-transformed mouse cells. It was found that these cells contain about six copies of a segment of DNA which includes the early region of the SV40 genome, and about one copy of the late viral sequences. To map the region of the viral genome which is transcribed in SVT2 cells, separated strands of each of the four fragments were prepared and hybridized to total transformed cell RNA. Part of the E strands of the two DNA fragments (A and C) which span the early region of the SV40 genome were found to enter the hybrid.

Animals↗
PubMed1974

Infection of EBV-genome-negative and -positive human lymphoblastoid cell lines with biologically different preparations of EBV.

Epstein-Barr virus (EBV) derived from the B95-8 line has transforming activity for cord blood cells, whereas virus derived from the P3HR-1 line lacks such activity. When the two viral preparations were compared for their ability to infect the same EBV-genome-negative lymphoblastoid cell line, BJA-B, they induced approximately the same number of EBV-determined nuclear antigen (EBNA)-positive cells. EBNA is compatibile with continued cell proliferation. No early antigen (EA)-positive cells appeared in the B95-8 virus-infected cultures, whereas P3HR-1 virus-infected cells went on to express EA. EA signals the entry of the cell into the lytic cycle. No late viral antigen (VCA) appeared and, as a consequence, the P3HR-1 virus infection became abortive. In contrast, the EBNA-positive cells induced by the B95-8 virus continued to divide over several weeks. These findings show that different EBV isolates may differ in their biological activity, probably due to their having different degrees of viral dependence on restrictive host cell controls.

Antigens, Viral↗
PubMed1973

Transcription of simian virus 40. 3. Mapping of "early" and "late" species of RNA.

To determine the orientation of transcription of the E and L strands of DNA from simian virus 40 (SV40), we used linear DNA prepared by cleavage of superhelical viral DNA by endonuclease R.R(1) from Escherichia coli as a primer.template for DNA polymerase. The resulting molecules, which were labeled only at the 3' end of each DNA strand, were then cleaved with Hemophilus parainfluenzae endonuclease Hpa I. The ensuing four DNA fragments, whose locations on the viral genome are known, were separated by electrophoresis, denatured, and hybridized to asymmetric SV40 complementary RNA. From the pattern of hybridization of the fragments containing the labeled 3' ends, we conclude that transcription of SV40 proceeds in a clockwise direction on the L strand and in a counterclockwise direction on the E strand as drawn on the conventional SV40 map. To map the "early" and "late" regions of the viral genome, we extracted RNA from lytically infected cells and hybridized it to the separated strands of the four fragments of (32)P-labeled SV40 DNA. Early after infection, RNA complementary to part of the E strand of the contiguous fragments A and C was detected. Late polysomal RNA was complementary to part of the L strand sequences of fragments A and C and to the total L strand sequence of fragments B and D.

Animals↗
PubMedDate not supplied

EBNA-1: a protein pivotal to latent infection by Epstein-Barr virus.

Epstein-Barr nuclear antigen 1, or EBNA-1, is required for the replication of the EBV genome as an extra-chromosomal element and is a key transcriptional regulator of this virus's latent gene expression. In this review we will describe the salient features of EBNA-1 and oriP, the latent origin of EBV to which EBNA-1 binds site-specifically. EBNA-1's association with host cellular factors, its association with metaphase chromosomes, and its ability to link DNAs to which it binds will be discussed in relation to its roles in replication and transcriptional activation. Although the mechanisms by which EBNA-1 facilitates replication and transcription largely remain enigmatic, EBV's viral replicon has been exploited successfully for applications in gene therapy and in the design of eukaryotic vectors for use in cell culture.

Animals↗
PubMedDate not supplied

Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells.

Epstein-Barr virus (EBV) infects human B lymphocytes, transforming the infected cells into dividing blasts that can proliferate indefinitely. The viral genome of 172 kilobase pairs (kbp) is a plasmid in most transformed cells. We have identified a region of EBV DNA, termed oriP (nucleotides 7,333-9,109 of strain B95-8), which acts in cis to permit linked DNAs to replicate as plasmids in cells containing EBV DNA. We have postulated the existence of a trans-acting gene allowing oriP function. Here we report that this gene lies in a 2.6-kbp region of the viral genome (nucleotides 107, 567-110, 176) which encodes the EBNA-1 antigen. We show that circular DNAs containing oriP, the EBNA-1 gene and a selectable marker replicate autonomously in cells derived from at least four developmental lineages and from at least three species. We also find that the one-third of the EBNA-1 gene repetitive in sequence is not essential for the trans-acting function that EBNA-1 gives oriP.

Animals↗
PubMedDate not supplied

Epstein-Barr virus: a human pathogen inducing lymphoproliferation in vivo and in vitro.

Epstein-Barr virus (EBV) is a pathogen that is associated with several diseases in humans. It causes most heterophile-positive cases of infectious mononucleosis. The virus is associated with another lymphoproliferative disease, African Burkitt's lymphoma, and with a malignancy of the nasopharynx, nasopharyngeal carcinoma. EBV appears to be a causative agent for some fatal lymphoproliferations in congenitally immunodeficient people. One proliferating cell type in infectious mononucleosis is an EBV-infected B lymphoblast. The tumor cell in African Burkitt's lymphoma is also an EBV-infected B lymphoblast. In vitro, EBV induces and maintains blast transformation of human B lymphocytes. All EBV-transformed cells, whether infected in vivo or in vitro, share many (but not all) virus-associated characteristics. Studies can now be performed in vitro that permit analysis of the various EBV-transformed cells and the host's immune responses to them.

Antibodies, Heterophile↗