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T Kouzarides

Publications and source records attributed to T Kouzarides.

88 records · Page 5Linked to original sources
PubMed1991

The DNA sequence of the human cytomegalovirus genome.

In the first part of this article we review what has been learnt from the analysis of the sequence of HCMV. A summary of this information is presented in the form of an updated map of the viral genome. HCMV is representative of a major lineage of herpesviruses distinct from previously sequenced members of this viral family and demonstrates striking differences in genetic content and organization. The virus encodes approximately 200 genes, including nine gene families, a large number of glycoprotein genes, and homologues of the human HLA class I and G protein-coupled receptor genes. The HCMV sequence thus provides a sound basis for future molecular studies of this highly complex eukaryotic virus. The second part discusses the practical rate of DNA sequencing as deduced from this and other studies. The 229 kilobase pair DNA genome of human cytomegalovirus (HCMV) strain AD169 is the largest contiguous sequence determined to date, and as such provides a realistic benchmark for assessing the practical rate of DNA sequencing as opposed to theoretical calculations which are usually much greater. The sequence was determined manually and we assess the impact of new developments in DNA sequencing.

Base Sequence↗
PubMed1991

The BZLF1 protein of EBV has a coiled coil dimerisation domain without a heptad leucine repeat but with homology to the C/EBP leucine zipper.

The EBV transactivator protein BZLF1 can bind many sites in the EBV genome, most of which have homology to a consensus AP-1 site, the binding site for the fos/jun family of transcription factors. Here we present evidence that BZLF1 can also recognise the binding site for the CCAAT/enhancer binding protein C/EBP and that a BZLF1 binding site within the BZLF1 promoter is recognised by the C/EBP protein. Analysis of the BZLF1 DNA binding domain suggests that the BZLF1 protein binds to DNA as a dimer using sequences adjacent to a basic DNA binding motif. The BZLF1 dimerisation domain does not have a heptad repeat of leucine residues common to leucine zipper proteins but does have characteristics of a coiled coil structure, as judged by site directed mutagenesis. We therefore propose that the dimerisation domain of BZLF1 is structurally related to the coiled coil structure of leucine zippers but lacks the highly conserved leucine repeat. We show that the PZLF1 dimerisation domain has residues in common with the C/EBP leucine zipper and discuss the possible implications of this relationship.

Amino Acid Sequence↗
PubMed1991

In vitro DNA binding activity of Fos/Jun and BZLF1 but not C/EBP is affected by redox changes.

The leucine zipper family of proteins have a DNA binding domain composed of a leucine zipper dimerisation interface and a basic DNA binding structure. We show here that redox changes affect the in vitro DNA binding ability of a select subset of leucine zipper proteins. The bacterially expressed DNA binding domains of Fos/Jun and BZLF1 are unable to bind DNA under non-reducing conditions whereas binding of the C/EBP DNA binding domain is unaffected. Sensitivity to redox state is due to the presence of a conserved cysteine residue in the basic DNA binding motif of Fos, Jun and BZLF1 but not C/EBP. Under non-reducing conditions an intermolecular disulphide bridge is formed between the cysteine residues of each basic motif within a dimer, which prevents DNA binding. We show that oxidation of these C residues can be achieved enzymatically, using glutathione peroxidase, and that DNA binding protects them from oxidation. These data raise the possibility that intracellular changes in the redox state may differentially regulate the activity of leucine zipper family members. In addition the loss of DNA binding activity under non-reducing conditions has implications for the purification methods used to isolate proteins of the leucine zipper family for structural analysis.

Amino Acid Sequence↗
PubMed1989

Leucine zippers of fos, jun and GCN4 dictate dimerization specificity and thereby control DNA binding.

The products of the fos and jun protooncogenes form a stable heterodimer which binds to the TPA-responsive element (TRE) TGACTCA with high affinity. These two proteins, together with the yeast GCN4 protein, belong to a growing family of transcription factors, including FosB, Fra1, JunB and JunD, whose members share a highly conserved DNA-binding domain. This domain is composed of two structures: a basic motif, which is thought to bind directly to DNA; and a leucine zipper, which provides a dimerization interface. Although this domain is highly conserved in Fos, Jun and GCN4, each of these three proteins has very different relative affinities for the TRE. To understand these differences, we used 'domain-swapping' experiments designed to test the relative contributions of the basic motif and the leucine zipper to TRE-binding affinity. Here we show that fos, jun and GCN4 have different affinities for the TRE due to differences in the hetero- or homo-dimerization capacity of their leucine zipper domains; the basic motifs of these three proteins have comparable DNA binding potential. These results indicate that leucine zippers control the types of protein complexes which can associate with a TRE and regulate gene expression.

DNA↗
PubMed1989

Epstein-Barr virus BZLF1 trans-activator specifically binds to a consensus AP-1 site and is related to c-fos.

Two regions of the Epstein-Barr virus BZLF1 trans-activator protein have sequence similarity to the c-fos protein. Part of the similarity corresponds to the region of c-fos which is similar to the DNA binding domain of c-jun and GCN-4. The structure of the exon which contains this region in c-fos and BZLF1 is also highly conserved between the two genes. Complete BZLF1 protein and a C terminal fragment were prepared either as purified fusion proteins or by in vitro translation from a BZLF1 cDNA. Gel retardation and DNase footprinting assays using these proteins show that BZLF1 is a sequence specific DNA binding protein capable of binding to a target sequence which contains a consensus AP-1 site.

Amino Acid Sequence↗
PubMed1989

Behind the Fos and Jun leucine zipper.

The production of the nuclear oncoproteins Fos and Jun is rapidly induced in response to extracellular signals. In the nucleus, the two proteins combine to form a tight complex via leucine zipper domains. The resulting Fos-Jun heterodimer can bind to the TPA-responsive element (TRE) by way of a novel, highly basic motif and can activate the transcription of TPA-responsive genes. The existence of several Fos- and Jun-related proteins with dimerization and DNA binding properties similar to Fos and Jun suggests that these two oncoproteins may be part of a network of related but functionally distinct transcription factors.

Amino Acid Sequence↗
PubMed1988

The role of the leucine zipper in the fos-jun interaction.

Mutagenesis of the fos protein supports the hypothesis that a heptad repeat of leucine residues stabilizes the interaction between the fos and jun proteins. We show that the complex between fos and jun can bind to DNA more tightly than either protein alone and that basic residues adjacent to the leucine repeat of fos contribute to the DNA-binding potential of the complex.

Animals↗
PubMed1988

An immediate early gene of human cytomegalovirus encodes a potential membrane glycoprotein.

The sequence of a region of the HCMV genome transcribed during the immediate early (IE) transcriptional phase has been determined. Transcription analysis of this region at the junction between fragments HindIII Z and J has identified three moderately abundant mRNAs of 3.4, 1.7, and 1.65 kb. The 3.4-kb RNA is expressed only under IE conditions of infection. It is composed of four exons and its predicted translation product has features characteristic of a membrane-bound glycoprotein. The 1.7-kb RNA is transcribed at both IE and late times postinfection. It is expressed from the same promoter as the 3.4-kb RNA and does not appear to be spliced. The 1.65-kb RNA is present at the IE phase, but is more abundantly transcribed at late times. It is composed of two exons and is 3' coterminal to the 3.4-kb RNA. Within the predicted translation product of the 1.65-kb RNA there are three regions which show homology to a family of related open reading frames found within the short unique region of HCMV.

Amino Acid Sequence↗
PubMed1987

Large-scale rearrangement of homologous regions in the genomes of HCMV and EBV.

The 20,349-bp sequence of the human cytomegalovirus (HCMV) HindIII F fragment has revealed eight open reading frames with homology to herpes simplex virus (HSV) and/or Epstein-Barr virus (EBV). With respect to EBV, these homologous genes can be divided into two blocks: one block contains three genes, including the DNA polymerase and glycoprotein B, and the other block contains five genes of unknown function. Although the relative organisation of genes within each block is identical in HCMV and EBV, the relative position of each block within the two genomes differs: in HCMV the two blocks are present directly adjacent to each other, whereas in EBV they are found 92 kb apart. This suggests that a genetic rearrangement has occurred in this region. Transcription analysis of the glycoprotein B gene is presented and the evolutionary relationship between the genomes of HCMV, EBV, and HSV is discussed.

Amino Acid Sequence↗
PubMed1987

Sequence and transcription analysis of the human cytomegalovirus DNA polymerase gene.

DNA sequence analysis has revealed that the gene coding for the human cytomegalovirus (HCMV) DNA polymerase is present within the long unique region of the virus genome. Identification is based on extensive amino acid homology between the predicted HCMV open reading frame HFLF2 and the DNA polymerase of herpes simplex virus type 1. We present here a 5280-base-pair DNA sequence containing the HCMV pol gene, along with the analysis of transcripts encoded within this region. Since HCMV pol also shows homology to the predicted Epstein-Barr virus pol, we were able to analyze the extent of homology between the DNA polymerases of three distantly related herpesviruses, HCMV, Epstein-Barr virus, and herpes simplex virus. The comparison shows that these DNA polymerases exhibit considerable amino acid homology and highlights a number of highly conserved regions; two such regions show homology to sequences within the adenovirus type 2 DNA polymerase. The HCMV pol gene is flanked by open reading frames with homology to those of other herpesviruses; upstream, there is a reading frame homologous to the glycoprotein B gene of herpes simplex virus type 1 and Epstein-Barr virus, and downstream there is a reading frame homologous to BFLF2 of Epstein-Barr virus.

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↗
PubMed1986

Identification of the human cytomegalovirus glycoprotein B gene and induction of neutralizing antibodies via its expression in recombinant vaccinia virus.

A human cytomegalovirus (HCMV) glycoprotein gene with homology to glycoprotein B (gB) of herpes simplex virus and Epstein-Barr virus and gpII of varicella zoster virus has been identified by nucleotide sequencing. The gene has been expressed in recombinant vaccinia virus and the gene product recognized by monoclonal antibodies and human immune sera. Rabbits immunized with the recombinant vaccinia virus produced antibodies that immunoprecipitate gB from HCMV-infected cells and neutralize HCMV infectivity in vitro. These data demonstrate a role for this protein in future HCMV vaccines.

Amino Acid Sequence↗
PubMed1983

Nucleotide sequence of the transforming region of human cytomegalovirus.

We report the nucleotide sequence of a BamHI-HindIII fragment of human cytomegalovirus containing the region (XbaI-HindIII) capable of transforming NIH3T3 cells. The single EcoRI site, shown to abolish transforming activity if cleaved, is present within an 8 base-pair inverted complementary repeat. Close to this sequence there is a small, potentially spliced, open reading frame possessing some of the signals involved in eukaryotic gene expression. Possible mechanisms of transformation, involving the inverted repeat sequence, are discussed.

Base Sequence↗
PubMedDate not supplied

The CBP co-activator is a histone acetyltransferase.

The CBP protein acts as a transcriptional adaptor for many different transcription factors by directly contacting DNA-bound activators. One mechanism by which CBP is thought to stimulate transcription is by recruiting the histone acetyltransferase (HAT) P/CAF to the promoter. Here we show that CBP has intrinsic HAT activity. The HAT domain of CBP is adjacent to the binding site for the transcriptional activator E1A. Although E1A displaces P/CAF from CBP, it does not disrupt the CBP-associated HAT activity. Thus E1A carries HAT activity when complexed with CBP. Targeting CBP-associated HAT activity to specific promoters may therefore be a mechanism by which E1A acts as a transcriptional activator.

Acetyltransferases↗