Biomedical subjects

P J Farrell

Publications and source records attributed to P J Farrell.

At least 91 records · Page 5Linked to original sources

PubMed1989

A new and sensitive method of screening for human papillomavirus infection.

Human papillomaviruses (HPVs) have been implicated in the development of cervical cancer, and HPV screening may one day supplement cytologic analysis of cervical samples. Detection of the small amounts of HPV DNA in cervical scrapes has been very difficult. The polymerase chain reaction is a new technique that can specifically amplify target DNA to facilitate its detection. We have amplified a region of HPV type 16 DNA using this technique and have shown the method to be both specific and sensitive (a tenfold improvement when compared with Southern blotting). Among 21 cervical scrapes, five from women with normal smears and 16 from women with dyskaryotic smears, HPV 16 DNA was present in scrapes from two women (40%) with normal smears and in scrapes from 12 women (75%) with dyskaryotic smears. The polymerase chain reaction technique is rapid, requires no radioactive probes, and should be the method of choice in future studies to determine the prevalence of HPV infection of the cervix.

DNA, Viral↗

PubMed1988

A spliced Epstein-Barr virus gene expressed in immortalized lymphocytes is created by circularization of the linear viral genome.

Epstein-Barr virus (EBV) immortalizes B-lymphocytes efficiently in vitro and in most or all of the cells a latent infection is established. Only a few viral genes are expressed during latency and these apparently maintain cell immortalization by EBV. A 1.9 kb cDNA clone of an EBV mRNA has been isolated from a B95-8 cDNA library and this cDNA corresponds to a new member of the group of EBV latent cycle genes. On Northern blots two polyadenylated transcripts 1.7 and 2.0 kb in length have been detected by the cDNA clone in B95-8 cells as well as in latently infected cell lines and in various cell lines derived from Burkitt's lymphomas. The cDNA sequence showed that this gene consists of nine exons and showed that this gene consists of nine exons and that the transcription unit crosses the terminal repeats. Thus, the intact gene is created upon infection by the circularization of the linear viral DNA molecule at the terminal repeats. One major open reading frame is present in the cDNA apparently coding for a 53 kd protein. A strongly hydrophilic part of the protein is followed by a very hydrophobic C-terminus, perhaps suggesting a membrane protein. The gene is unrelated to the other latent cycle genes, the EBV nuclear antigens (EBNA 1-4), the leader protein (LP), and the latent membrane protein (LMP) genes. This is the first example of a viral gene whose coding sequence is only created by joining of the ends of a linear virus.

Amino Acid Sequence↗

PubMed1988

Sequence analysis of Raji Epstein-Barr virus DNA.

The DNA sequence of the EcoRI Dhet and part of the BamHI E fragments of Raji EBV has been determined. Precise locations of two deletions in Raji DNA have been identified and their consequences for gene structure evaluated. The deletion in Raji of reading frames BALF1, BARF1, and BZLF2 and truncation of BALF2 and BERF5 probably account for the replication defect in this strain. The degree of sequence variation between B95-8 and Raji has been examined and shows considerable variation between genes. The latent membrane protein gene is exceptionally polymorphic and the initiator methionine for the late productive cycle protein overlapping the latent membrane protein is absent in Raji.

Amino Acid Sequence↗

PubMed1987

Differences in B cell growth phenotype reflect novel patterns of Epstein-Barr virus latent gene expression in Burkitt's lymphoma cells.

Recently established Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) cell lines, carrying chromosomal translocations indicative of their malignant origin, have been monitored for their degree of in vitro progression towards a more 'lymphoblastoid' cell surface phenotype and growth pattern, and for their expression of three EBV latent gene products which are constitutively present in all virus-transformed normal lymphoblastoid cell lines (LCLs). BL cell lines which stably retained the original tumour biopsy phenotype on serial passage were all positive for the nuclear antigen EBNA 1 but did not express detectable amounts of two other 'transforming' proteins, EBNA 2 and the latent membrane protein (LMP). This novel pattern of EBV gene expression was also observed on direct analysis of BL biopsy tissue. All three viral proteins became detectable, however, in BL cell lines which had progressed towards a more LCL-like phenotype in vitro. This work establishes a link between B cell phenotype and the accompanying pattern of EBV latent gene expression, and identifies a novel type of EBV:cell interaction which may be unique to BL cells.

Antibodies, Monoclonal↗

PubMed1987

Novel nuclear antigens recognized by human sera in lymphocytes latently infected by Epstein-Barr virus.

We have used three latently infected cell lines, X50-7, JC-5, and Raji, to identify two new nuclear antigen complexes by Western immunoblotting with human anti-EBNA (Epstein-Barr Virus Nuclear Antigen) sera. One antigen complex, termed EBNA III, is composed of a group of high molecular weight proteins between 130 and 160 kDa and the other antigen complex, termed EBNA IV, is a size-related group of polypeptides between 28 and 62 kDa. Both the EBNA III and EBNA IV groups of proteins display variation in size among the different strains of EBV. Cell fractionation of X50-7, JC-5, Raji, and C16, a cell clone of P3HR1, showed that both new antigen complexes were completely recovered from the nuclei of latently infected lymphocytes as were previously described EBNA I and II. Because these new antigens are only detected by anti-EBNA sera in EBV infected cells, it seems likely that they may be encoded by the viral genome and play some role in the immortalization of lymphocytes by the virus.

Antigens, Viral↗

PubMed1987

Mapping of the gene coding for Epstein-Barr virus-determined nuclear antigen EBNA3 and its transient overexpression in a human cell line by using an adenovirus expression vector.

The open reading frame which lies within the Epstein-Barr virus (EBV) T2 cDNA isolated by Bodescot et al. (M. Bodescot, O. Brison, and M. Perricaudet, Nucleic Acids Res. 14:2611-2620, 1986) was inserted into a eucaryotic expression vector containing a strong adenovirus promoter. The T2 cDNA contains viral genomic sequences from the short BLRF3 open reading frame fused to the adjacent BERF1 long open reading frame. After transfection of human cells, the recombinant plasmid directed the expression of a 140-kilodalton protein. The expressed protein had the same molecular weight, subcellular localization, and immunological characteristics as the EBV-determined nuclear antigen EBNA3, which is made in lymphocytes latently infected with EBV. Immunoprecipitation of extracts of transfected cells labeled with [32P]phosphoric acid showed that the EBNA3 protein is phosphorylated.

Adenoviruses, Human↗

PubMed1987

A promoter for the highly spliced EBNA family of RNAs of Epstein-Barr virus.

A transcription start for the highly spliced EBNA group of RNAs in B95-8 cells has been identified in the short unique region of the virus genome. This promoter is used in many (but not all) human cell lines carrying Epstein-Barr virus, including a tightly latent human lymphoblastoid cell line. Another promoter for the EBNA RNAs was described previously in the internal repeat region of the virus genome. The existence of these alternative promoters may be important for differential control of EBNA gene expression.

Antigens, Viral↗

PubMed1987

Sequences of the Epstein-Barr Virus (EBV) large internal repeat form the center of a 16-kilobase-pair palindrome of EBV (P3HR-1) heterogeneous DNA.

We have previously characterized several genomic rearrangements of Epstein-Barr virus (EBV) DNA contained in one of the defective EBV genomes harbored by the P3HR-1 (HR-1) line (H. B. Jenson, M. S. Rabson, and G. Miller, J. Virol. 58:475-486, 1986). One recombinant clone of heterogeneous DNA (het DNA) from this defective genome is an EcoRI fragment of 16 kilobase pairs (kbp) which is a palindrome. DNA digestion fragments specific for the center of this palindrome were present in cells which contained het DNA but not in cells which lacked het DNA. Thus, the palindrome was not an artifact of DNA cloning. The organization of the center of this palindrome was studied by DNA sequencing. The comparable region of the parental HR-1 genome was also studied by DNA sequencing. The central 3,495 base pairs (bp) of the palindrome were composed of sequences derived exclusively from internal repeat 1 of EBV, represented by BamHI W fragment. At each end of the central 3,495 hp was a symmetrical recombination with sequences of BamHI-Z, located more than 50 kbp away on the standard EBV genome. The central 3,495 bp were composed of an unduplicated 341 bp flanked by two perfect palindromic repeats of 1,577 bp. The 341-bp unique region was a portion of a 387-bp region of standard HR-1 BamHI-W which was identical to the central 387 bp of the palindrome. This central 387-bp region contained numerous stretches of dyad symmetry capable of forming a large stem-and-loop structure. The palindromic rearrangement had created two novel open reading frames in het DNA derived from standard HR-1 BamHI-W sequences. These two het DNA open reading frames had different amino termini but identical carboxy termini derived from the large open reading frame in standard HR-1 BamHI-W (HR-1 BWRF1). The BamHI-W sequences found in het DNA did not include either the TATA box of standard HR-1 BamHI-W or the exons which are present in the potentially polycistronic latent mRNAs encoding EBV nuclear antigens. These marked alterations in genomic structure may relate to the unique biologic properties of virus stocks containing het DNA by creation of new polypeptides or by formation or deletion of regulatory or functional signals.

Base Sequence↗

PubMed1986

Coding content and expression of the EBV B95-8 genome in the region from base 62,248 to base 82,920.

The DNA sequence of a 20,672-base region of the EBV B95-8 genome from the BamHI site separating fragments BamHI-F and BamHI-Q to the EcoRI site separating fragments EcoRI-G2 and EcoR1-F has been determined. S1 mapping and northern blotting with probes from M13 recombinants have been used to search for RNAs from this region. Five mRNAs have been identified and correlated with five open reading frames in the DNA sequence. Two of these open reading frames encode ribonucleotide reductase subunits. A further two open reading frames are present including one for a predicted protein of 340 kDa for which no transcript has yet been detected.

Base Sequence↗

PubMed1986

Restricted expression of EBV latent genes and T-lymphocyte-detected membrane antigen in Burkitt's lymphoma cells.

Certain newly established Epstein-Barr virus-containing Burkitt's lymphoma cell lines do not express the cytotoxic T-lymphocyte-detected membrane antigen (LYDMA) through which EBV infection is normally controlled by the host. When the EB virus recovered from these BL lines was used to transform peripheral blood lymphocytes from seronegative donors, the lymphoblastoid cell lines (LCLs) that arose were all LYDMA positive. This indicates that the LYDMA-negative nature of the BLs is not the result of a mutation in the resident viral genome but is rather a specific adaptation in those cells, perhaps permitting evasion of the host immune surveillance in tumour development. A comparison of the EBV gene expression in six LYDMA-negative and two LYDMA-positive BL lines and in their corresponding LCLs revealed that several of the BL lines did not express all of the viral gene products classically associated with latent transformation by EBV. Four out of eight cell lines showed restricted expression of the latent membrane protein (LMP) and/or the EB nuclear antigen, EBNA 2. A new level of EBV gene regulation therefore appears to be operating in some of the BL cell lines. The patterns of expression of EBV genes in the cell lines did not show any correlation with the known susceptibility of the lines to T cell killing.

Antigens, Surface↗

PubMed1985

Prevalence of human papillomavirus type 16 DNA in cervical carcinoma samples in East Anglia.

A substantial increase in the incidence of severely dysplastic cervical lesions (CIN 3) has been observed during the period 1975-1982 in the East Anglian region of England. Since patients with severe dysplasia have an enhanced risk of developing cervical carcinoma, it seems possible that a substantial increase in the rate of cervical carcinoma is likely to occur in the near future. Evidence of a relationship between human papillomavirus (HPV) infection and cervical carcinoma has accumulated recently. We have studied the incidence of HPV16 DNA in cervical tissue samples from patients with cervical carcinoma, severe dysplasias and normal controls. Five out of 11 invasive squamous carcinomas of the cervix, 3/4 dysplasias and 0/12 normal samples were positive in Southern blot assays for HPV16 DNA. Some of the tissue samples had as many as 500 copies of HPV16 DNA per cell. The amount of HPV16 DNA present correlated with the aggressiveness of tumour growth.

Animals↗

PubMed1985

Chromatin structure of Epstein-Barr virus.

The episomal copies of Epstein-Barr virus (EBV) DNA are in a chromatin structure in the lymphoblastoid cell line B95-8. Nucleosomes on EBV DNA have the same spacing as those in cellular chromatin. Some of the EBV DNA is not in nucleosomes; this probably corresponds to DNA which is being packaged. Several DNase hypersensitive sites have been mapped on the EBV genome and patterns of CpG methylation are examined.

Animals↗

PubMed1985

Two related but differentially expressed potential membrane proteins encoded by the EcoRI Dhet region of Epstein-Barr virus B95-8.

Three mRNAs in the EcoRI Dhet region of Epstein-Barr virus B95-8 were mapped. Their 3' ends are coterminal. A latent gene containing three exons is transcribed from the ED-L1 promoter and was predicted to lead to expression of a 42-kilodalton protein. An unspliced late mRNA is produced by transcription from the ED-L1A promoter within the first intron of the above gene and was predicted to lead to expression of a 28-kilodalton protein corresponding to the C-terminal two-thirds of the 42-kilodalton protein. Both proteins would have hydrophobic N-terminal domains and highly acidic C-terminal domains and were predicted to span the cell membrane. An early promoter (ED-L2) is located in the 3' untranslated region of the above genes and was predicted to lead to expression of a 6.5-kilodalton polypeptide containing a C-terminal hydrophobic region.

Base Sequence↗

PubMed1984

Transcription and DNA sequence of the BamHI L fragment of B95-8 Epstein-Barr virus.

The sequence of the BamHI L fragment of B95-8 Epstein-Barr virus (EBV) DNA has been determined. The transcription starts of five promoters have been mapped to this fragment, using S1 mapping and either in vitro transcription or the primer extension technique. Dramatically increased levels of cytoplasmic poly(A) + RNAs, transcribed from these promoters, occur after treatment of B95-8 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA). For three of these promoters addition of phosphonoacetic acid (PAA) inhibits the effect of TPA, indicating that they give rise to late lytic cycle RNAs. The other two promoters give rise to early RNAs. Northern blot analysis indicates that one of the late promoters initiates two transcripts whose size differences are due to different splicing patterns. These two RNAs code for the 350/300 and 250/200 kd envelope glycoproteins of EBV. The sequences of these proteins would be of use in the production of a synthetic vaccine to prevent EBV infection.

Animals↗

PubMed1983

Latent and lytic cycle promoters of Epstein-Barr virus.

Four RNA polymerase II promoters have been mapped in the DNA sequence of the EcoRI-H and -Dhet fragments of B95-8 Epstein-Barr virus. RNAs transcribed from three of these promoters are dramatically induced by treatment of B95-8 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA). The other promoter is active with or without TPA treatment of the cells and is thus active in the latent virus cycle. Deletion mapping suggests that DNA sequence homologies between some of the promoters lie in the same region as essential upstream promoter elements.

Base Sequence↗

PubMed1983

Homologous upstream sequences near Epstein-Barr virus promoters.

The sequence of the 17,166-base-pair EcoRI C fragment of Epstein-Barr virus DNA, cell line B95-8, was determined. In vitro transcription was used to identify three RNA polymerase II promoters within this fragment of the virus. Cytoplasmic poly(A)+ RNAs starting at these points were demonstrated in B95-8 cells induced into virus production with 12-O-tetradecanoylphorbol 13-acetate. Uninduced B95-8 cells contained much less of these RNAs. The upstream sequences near the three promoters show striking homologies that may be involved in transcriptional control.

Base Sequence↗

PubMed1983

Sequence analysis of the 17,166 base-pair EcoRI fragment C of B95-8 Epstein-Barr virus.

In order to provide a framework for understanding the molecular biology of Epstein-Barr virus (EBV), we are determining the DNA sequence of the virus and studying the organization of genes on the viral genome. In this paper we report the DNA sequence of the EcoRI C fragment of the B95-8 strain of EBV. The large (approximately 13.6 kb) deletion in this strain has been located by comparison with the DNA sequence of EBV isolated from Raji cells. The sequence has been analysed for possible protein coding regions and transcriptional control sites. At least eight large open reading frames are found, some of them associated with canonical promoter and polyadenylation sequences. The sequences of some of the encoded proteins suggest that they are membrane proteins. It is known that antibodies to major membrane glycoproteins of EBV can neutralize infection in tissue culture. A possible relationship between some of the encoded proteins and the major membrane glycoproteins of the virus is discussed.

Base Sequence↗

PubMed1983

DNA sequence and transcription of the BamHI fragment B region of B95-8 Epstein-Barr virus.

The DNA sequence of the BamHI fragments B and the contiguous portion of G extending from the BamHI site to the EcoRI site of the B95-8 strain of Epstein-Barr virus has been determined. The sequence has been analysed for possible protein coding regions and transcriptional control sites. At least eight large open reading frames were found. Several RNA polymerase II promoters have been identified in the BamHI-B fragments. RNAs transcribed from these promoters are substantially induced by treatment of B95-8 cells with 12-O-tetradecanoylphorbol-13-acetate. Phosphonoacetic acid reversed the stimulatory effect of 12-O-tetradecanoylphorbol-13-acetate on the transcription of these RNAs indicating that they are late RNAs.

Base Sequence↗