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Biomedical subjects

B Fleckenstein

Publications and source records attributed to B Fleckenstein.

At least 145 records · Page 8Linked to original sources
PubMed1988

Thymidylate synthase gene of herpesvirus ateles.

The putative thymidylate synthase (TS) gene of herpesvirus ateles, a T-lymphotropic tumor virus of New World primates, has a single large open reading frame encoding a polypeptide of 32.9 kilodaltons. The gene is transcribed into an unspliced 2.4-kilobase mRNA that is abundantly expressed late in virus replication. The AT-rich 5' untranslated leader sequence of TS mRNA in herpesvirus ateles-infected cells is remarkable in length (1,184 nucleotides), containing 29 minicistrons; this may indicate a role in translation regulation.

Amino Acid Sequence↗
PubMed1987

[Serologic AIDS diagnosis with polypeptides obtained by genetic technics of the human immunodeficiency virus (HIV-1)].

Serologic testing for human immunodeficiency virus type 1 (HIV-1) is currently based on enzyme linked immunosorbent assay (ELISA) as screening method. Positive ELISA-results have to be confirmed by at least one second procedure such as Western blotting or immunofluorescence. To obtain new diagnostic reagents for confirmatory testing, we expressed viral antigens in procaryotic systems. Peptides representing epitopes of structural core (gag)- and envelope (env)-proteins of HIV were produced in E. coli as stable immunogenic beta-galactosidase fusion proteins. Recombinant proteins were taken for immunoblot-assays. The results of Western blotting with those fusion proteins were in general comparable with conventional ELISA, immunofluorescence, immunoblot with cell-culture derived virus and commercially available ELISA tests based on recombinant proteins. Immunoblots using recombinant transmembrane protein (gp41) derived polypeptide were more sensitive than the conventional procedure with purified virion proteins. Western blotting with recombinant fusionproteins provide reliable and inexpensive serodiagnostics without handling of infectious cell cultures.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Binding of transcription factors and creation of a large nucleoprotein complex on the human cytomegalovirus enhancer.

The effect of the human cytomegalovirus immediate early region 1 enhancer on transcription was studied in vitro with HeLa cell nuclear extract. Stimulation of in vitro transcription mediated by the enhancer element involves its recognition by specific trans-acting factors present in the nuclear extract. DNase I protection analysis was used to determine at the nucleotide level those enhancer sequences that interact with nuclear factors. At least nine sites of protein-DNA interaction were detected over approximately 400 base pairs of enhancer sequence. The regions of nuclease protection are associated with 21-, 19-, 18-, and 17-base-pair repeat elements as well as with a unique sequence, creating a large nucleoprotein complex. The relationship between the protein binding and the activity of the immediate early region 1 enhancer is discussed.

Base Sequence↗
PubMed1987

Organization of terminal reiterations in the virion DNA of herpesvirus saimiri.

The population of herpesvirus saimiri (HVS) genomes extracted from extracellular virions are double-stranded, linear DNA molecules of about 160 kilobase pairs (kbp) each composed of a central segment of 110 to 112 kbp and 36% (G + C) (i.e. 'light' or L-DNA) linked to direct reiterations of a 1.44 kbp repeat unit of 71% (G + C) (i.e. 'heavy' or H-DNA) at each terminus. In this paper, we show that the population of HVS DNA molecules contains approximately equal concentrations of genomes with all possible integral numbers of complete repeat units (i.e. from greater than 30 to 1) at either 'left' or 'right' ends but that all molecular ends are derived by a unique cleavage at a site close to the single ApaI restriction endonuclease site of the H-DNA repeat unit. Junctions of proximal H-DNA repeat units with L-DNA occur at, or very close to, the sequence present at the molecular ends. The transition from L- to H-DNA occurs abruptly at this site at the 'right' end of the L-DNA component but some rearranged restriction enzyme cleavage sites typical of H-DNA are found within the first 0.8 kbp of the L-DNA sequences at the 'left' H-L DNA junction. HVS appears to provide an extreme example of the general process whereby herpesvirus DNAs are matured from concatemeric intermediates by a site-specific cleavage/recombination process involving random choice between equivalent sites for the initiation of the process and with choices between alternative termination sites being limited by a headful packaging mechanism.

Base Composition↗
PubMed1987

The two major structural phosphoproteins (pp65 and pp150) of human cytomegalovirus and their antigenic properties.

Human cytomegalovirus (HCMV) purified from cell culture contains two dominant structural phosphoproteins with apparent molecular weights of 65,000 and 150,000, designated as pp65 and pp150 respectively. The humoral immune response of infected individuals against pp65 is relatively weak and is not always detectable by Western blot analyses. This report shows that recent clinical isolates of HCMV do not necessarily have pp65 as a prominent constituent, suggesting that the low immune reaction is due to variable expression of the pp65 in natural infections. However, the HCMV strains tested in this study produced the large structural phosphoprotein (pp150) in about equal amounts. The pp150 is remarkably immunogenic, if compared with all other virion constituents; serum pools and individual sera from HCMV-infected patients recognized this particular protein intensively in immunoblot assays. Thus, phosphoprotein pp150 seems to be the primary polypeptide candidate for expression cloning in order to develop reagents for novel ways of HCMV diagnosis.

Antibodies, Viral↗
PubMed1987

Genomic localization, sequence analysis, and transcription of the putative human cytomegalovirus DNA polymerase gene.

The human cytomegalovirus (HCMV)-induced DNA polymerase has been well characterized biochemically and functionally, but its genomic location has not yet been assigned. To identify the coding sequence, cross-hybridization with the herpes simplex virus type 1 (HSV-1) polymerase gene was used, as suggested by the close similarity of the herpes group virus-induced DNA polymerases to the HCMV DNA polymerase. A cosmid and plasmid library of the entire HCMV genome was screened with the BamHI Q fragment of HSV-1 at different stringency conditions. One PstI-HincII restriction fragment of 850 base pairs mapping within the EcoRI M fragment of HCMV cross-hybridized at Tm - 25 degrees C. Sequence analysis revealed one open reading frame spanning the entire sequence. The amino acid sequence showed a highly conserved domain of 133 amino acids shared with the HSV and putative Epstein-Barr virus polymerase sequences. This domain maps within the C-terminal part of the HSV polymerase gene, which has been suggested to contain part of the catalytic center of the enzyme. Transcription analysis revealed one 5.4-kilobase early transcript in the sense orientation with respect to the open reading frame identified. This transcript appears to code for the 140-kilodalton HCMV polymerase protein.

Amino Acid Sequence↗
PubMed1987

Primary structure and transcription of the genes coding for the two virion phosphoproteins pp65 and pp71 of human cytomegalovirus.

Human cytomegalovirus contains a phosphorylated matrix protein of 65,000 apparent molecular weight (65K phosphoprotein; pp65) and a related phosphoprotein of 71,000 molecular weight (pp71). The 65K phosphoprotein is usually by far the most abundant structural component found in culture-grown purified virus particles. This study describes the precise mapping of the genes for both polypeptides, giving the entire nucleotide sequences and the exact positions of the respective transcripts. The 65K phosphoprotein is coded for by the 5'-terminal part of an abundant 4-kilobase (kb) mRNA. The 71K phosphoprotein corresponds to the single translational reading frame of a rare nonspliced 1.9-kb mRNA that is coterminal with the 4-kb transcript. The promoter for 4-kb mRNA appears to be unusual in structure; it does not contain a characteristic TATA sequence. The expression of antigenic epitopes from pp65 may allow improved serodiagnosis of human cytomegalovirus infections.

Amino Acid Sequence↗
PubMed1987

Map position and nucleotide sequence of the gene for the large structural phosphoprotein of human cytomegalovirus.

Human cytomegalovirus particles contain a phosphoprotein of 150,000 (pp150) apparent molecular weight in their matrix; the protein appears particularly reactive in Western blot analyses with human antisera. The gene for pp150 was mapped by screening a bacteriophage lambda gt11 cDNA expression library with monospecific rabbit antisera. Subsequent hybridization of cDNA with cosmid and plasmid clones containing the human cytomegalovirus strain AD169 genome mapped the gene to HindIII fragments J and N. The gene is transcribed into a late 6.2-kilobase RNA. The nucleotide sequence of this region was determined, and a transcription initiation site and two polyadenylation sites of an abundant transcript were located by primer extension and nuclease protection experiments. The reading frame for pp150, deduced from computer analyses, gives rise to a polypeptide of 1,048 amino acids in length; protein secondary structure analysis revealed multiple beta-pleated sheets in hydrophilic clusters, providing a possible explanation for the immunogenic properties of the polypeptide.

Amino Acid Sequence↗
PubMed1987

The 160,000-Mr virion protein encoded at the right end of the herpesvirus saimiri genome is homologous to the 140,000-Mr membrane antigen encoded at the left end of the Epstein-Barr virus genome.

The sequence of 4.4 kilobase pairs (kbp) from the conventional right terminus of the A + T-rich light-DNA (L-DNA) sequences of the herpesvirus saimiri (HVS) genome contains a leftward-directed open reading frame (ORF) for a 1,299-residue protein. The molecular weight predicted for the protein (143,000) is in good agreement with the estimates of 150,000 to 160,000 for the major nonglycosylated polypeptide of the virion tegument (the 160K polypeptide), previously shown to be encoded by this region of the genome. The first initiation codon of the ORF is only 250 nucleotides from the junction of the L-DNA component with the G + C-rich terminal reiterations (i.e., heavy or H-DNA) of the genome. An unusually A + T-rich sequence (43 of 45 nucleotides are A or T, relative to a mean composition of 40% G + C for the ORF) occurs some 75 bp 5' to this initiation codon, and the first adenylation signal (AATAAA) on this DNA strand occurs 18 bp 3' to the termination codon. The amino acid sequence predicted for the 160K protein of HVS is homologous over most of its length to the 1,318-residue protein encoded by the leftmost major ORF of the G + C-rich genome of Epstein-Barr virus (BNRF1, the 140K nonglycosylated membrane antigen). No homology to either of these proteins is evident among the products predicted from the complete sequence of the alpha herpesvirus varicella-zoster virus. Thus gamma herpesviruses with coding sequences which differ in mean nucleotide composition by some 20% G + C have homologous proteins encoded at similar positions with respect to genome termini, with the right end of HVS being homologous to the left end of Epstein-Barr virus.

Amino Acid Sequence↗
PubMed1986

Nuclear factor 1 interacts with five DNA elements in the promoter region of the human cytomegalovirus major immediate early gene.

The human cytomegalovirus (HCMV), a ubiquitous pathogen of the herpesvirus group, has a linear double-stranded DNA genome of 235 kb. The expression of its major immediate early gene (IE1) is entirely dependent on host factors, presumably proteins binding to DNA elements in the regulatory regions of the gene. We have identified four high-affinity binding sites for nuclear factor 1 (NF1) in the promoter upstream region of IE1 gene between nucleotides -780 and -610, and an additional, even stronger, binding site in the first intron near nucleotide +350. NF1 activity is found in a wide range of species and binds to the sequence 5' TGGC/ANNNNNGCCAA3' on double-stranded DNA, protecting approximately 25 bp from DNase I digestion; its functional importance has been found first in the necessity for adenovirus DNA replication, where it may be important in mediating the binding of other proteins. The regulatory significance of NF1 recognition elements within other genes is unknown. The NF1 binding sites in the HCMV IE1 gene coincide with regions that had been shown to be sensitive to DNase I in the active gene but not sensitive in the silent gene; there was, however, no NF1 binding in the strong and constitutively DNase I-hypersensitive transcription enhancer of the IE1 gene. This suggests that the specific protein--DNA interaction described is important in the regulated control of the IE1 gene.

Animals↗
PubMed1986

The A+T-rich genome of Herpesvirus saimiri contains a highly conserved gene for thymidylate synthase.

Herpesvirus saimiri (HVS) is the prototype member of a distinctive subset of lymphotropic herpesviruses (the gamma 2 subgroup) with A+T-rich coding sequences. In this paper, we show that cells productively infected with HVS contain high concentrations of a virus-specified thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45); we identify the active polypeptide and present the sequence of the virus gene. The predicted amino acid sequence of the 294-residue subunit of the virus enzyme is 70% homologous with the sequence of the human enzyme and about 50% homologous with prokaryotic thymidylate synthases, illustrating the remarkable structural constraints imposed by the thymidylate synthase function. However, the presence of the enzyme is not a conserved property of herpesviruses. We find no evidence for a virus-encoded thymidylate synthase activity (or a homology to a thymidylate synthase sequence) in G+C-rich representatives of alpha 1 (e.g., herpes simplex viruses, 66-68% G+C), beta (i.e., human cytomegalovirus, 58-59% G+C), and gamma 1 (i.e., Epstein-Barr virus, 60% G+C) herpesvirus subgroups. The production of excess thymidylate by a virus thymidylate synthase in cells infected with an A+T-rich herpesvirus would provide one plausible source of biased mutations by the virus-encoded replicative enzymes, which we have previously suggested as the likely general cause of differences in the mean nucleotide compositions of herpesvirus genomes.

Amino Acid Sequence↗
PubMed1986

Mapping of the major glycoprotein gene of human cytomegalovirus.

The gene coding for the most abundant glycoprotein (gp58) of human cytomegalovirus (HCMV), strain AD169, was physically mapped on the viral genome. A monospecific rabbit antiserum against gp58 was used to screen a cDNA library that was constructed from poly(A)+ RNA of HCMV-infected cells in the prokaryotic expression vector lambda gt11. A cDNA clone was identified which synthesized part of the glycoprotein. It allowed localization of the coding region within the right terminal sequence of the HindIII-F fragment between map coordinates 0.344 and 0.380 of HCMV virion DNA.

Antibodies, Monoclonal↗
PubMed1986

Organization of the thymidylate synthase gene of herpesvirus saimiri.

Herpesvirus saimiri codes, unlike most other herpesviruses, for a thymidylate synthase (TS). The TS gene of herpesvirus saimiri is unusual in structure and regulation of expression. It is transcribed into a nonspliced mRNA of 2,190 nucleotides. The single open reading frame of the viral TS gene, instructing a polypeptide of 33.5 kilodaltons, has extensive sequence homology with the corresponding TS coding sequences of human cells and of various procaryotes; the putative polypeptide derived from the nucleotide sequence of the herpesvirus saimiri TS gene is 70% identical with the human enzyme. The untranslated regions of the herpesvirus saimiri TS gene do not share homology with the other characterized eucaryotic or bacterial TS genes. The 5' untranslated sequence has 22 ATG triplets shortly followed by stop codons. The herpesvirus saimiri TS gene, which may be weakly transcribed during immediate early and early times of virus replication, is maximally expressed at the late phase. Various parameters suggest that the TS gene has been acquired in virus evolution by an ancestral herpesvirus from the cellular genome.

Amino Acid Sequence↗
PubMed1985

Cytomegalovirus DNA in colorectal carcinoma tissues.

Numerous studies had linked human cytomegalovirus (HCMV) infections with neoplasia. Among various other malignant tumors, colonic carcinoma tissues were reported to contain DNA sequences hybridizing with DNA extracted from virus particles. Gene technology allowed us to use a cloned viral DNA library to measure HCMV in colorectal tumors more specifically. Four of 38 tissue specimens did contain DNA sequences homologous to cloned viral DNA probes; however, in each of those cases, identical hybridization patterns were seen with specimens from non-infiltrated surrounding intestinal wall. The amount of HCMV DNA in normal tissues was at least as much as in tumor biopsies. We conclude that nucleic acid hybridizations at high sensitivity with moleculary cloned HCMV DNA did not support the notion of a correlation between colorectal carcinomas and human cytomegalovirus.

Base Sequence↗
PubMed1985

A transcription enhancer in the Herpesvirus saimiri genome.

Herpesvirus saimiri, an oncogenic agent of New World primates, has a linear double-stranded DNA genome of approximately 155 kb. To test its genome for the presence of a transcription enhancer, we have mixed randomly fragmented H. saimiri DNA with non-infectious, linear SV40 DNA lacking the 72-bp repeat enhancer region (the so-called SV40 enhancer trap) and co-transfected this DNA mixture into monkey CV-1 cells. Viable SV40-like viruses were generated by intracellular ligation/repair processes with short H. saimiri DNA fragments. One recombinant, SVHS-2, had integrated a 377-bp enhancer segment from the righthand region of the H. saimiri genome, 7 kb upstream of DNA sequences encoding an immediate-early mRNA. This enhancer sequence is contained within the non-repetitive portions of the viral genome known to be preserved episomally in all lymphoid tumor cell lines. Further recombinant viruses (SVHS-14, SVHS-7, and SVHS-8) essentially contain subsets of the 377-bp insert. Unlike in the previous enhancer trap experiments, where heterologous enhancers were incorporated without any sequence alterations, SVHS-14 and SVHS-7 have suffered short internal deletions of a very similar segment of the H. saimiri insert. This renders the enhancer more active, implying that the deleted segment, while it may have a role in the herpesvirus infection cycle, exerts a negative effect within the isolated enhancer.

Antigens, Viral, Tumor↗
PubMed1985

A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus.

A strong transcription enhancer was identified in the genomic DNA (235 kb) of human cytomegalovirus (HCMV), a ubiquitous and severe pathogen of the herpesvirus group. Cotransfection of enhancerless SV40 DNA with randomly fragmented HCMV DNA yielded two SV40-HCMV recombinant viruses that had incorporated overlapping segments of HCMV DNA to substitute for the missing SV40 enhancer. Within HCMV, these enhancer sequences are located upstream of the transcription initiation site of the major immediate-early gene, between nucleotides -118 and -524. Deletion studies with the HCMV enhancer, which harbors a variety of repeated sequence motifs, show that different subsets of this enhancer can substitute for the SV40 enhancer. The HCMV enhancer, which seems to have little cell type or species preference, is severalfold more active than the SV40 enhancer. It is the strongest enhancer we have analyzed so far, a property that makes it a useful component of eukaryotic expression vectors.

Base Sequence↗
PubMed1985

Genome organization of herpesvirus aotus type 2.

Herpesvirus aotus type 2, a virus commonly found in owl monkeys without overt disease, has a similar genome structure to the oncogenic herpesviruses of nonhuman primates (herpesvirus saimiri, herpesvirus ateles). Virion DNA of herpesvirus aotus type 2 (M-DNA) has an unique 110-kilobase-pair region of low G + C content (40.2%, L-DNA), inserted between stretches of repetitive H-DNA (68.7% G + C, about 41 kilobase pairs per molecule) that are variable in length. A minority of virions contain defective genomes that consist of repetitive H-DNA only. The H-DNA is composed of various types of repeat units that are related in sequence with each other. The two dominant types of repeats (2.3 and 2.7 kilobase pairs) were cloned and compared by restriction enzyme cleavages and partial nucleotide sequencing. They are homologous in at least 1.3 kilobase pairs. The two forms of repeat units are randomly arranged and oriented in tandem. Reassociation kinetics did not allow detection of sequence homologies between H- and L-DNA of herpesvirus aotus type 2 and the respective sequences of oncogenic primate herpesviruses.

Animals↗