PubMed Health⌕ Search

Biomedical subjects

J E Mertz

Publications and source records attributed to J E Mertz.

At least 19 recordsLinked to original sources
PubMed2001

Nuclear factor 1 family members mediate repression of the BK virus late promoter.

BK virus (BKV) is a member of the polyoma virus family that is ubiquitous in humans. Its 5-kb DNA genome consists of a bidirectional promoter region situated between two temporally regulated coding regions. We mapped the transcription initiation site of the major late promoter (MLP) of the archetype strain BKV(WW) to nt 185. We found that it lies within the sequence TGGN6GCCA, a binding site for members of the nuclear factor 1 (NF1) family of transcription factors. Competition electrophoretic mobility shift and immunoshift assays confirmed that NF1 factors present in nuclear extracts of HeLa and CV-1 cells bind to the BKV-MLP. Because BKV(WW) grew poorly in tissue culture and failed to express detectable levels of RNA in vitro, SV40-BKV chimeric viruses were constructed to investigate the transcriptional function of this NF-1 binding site. These sequence-specific factors repressed transcription in a cell-free system when template copy number was low. This repression could be relieved by the addition in trans of oligonucleotides containing wild-type, but not mutated, NF1-binding site sequences. SV40-BKV chimeric viruses defective in this NF1-binding site overproduced late RNA at early, but not late, times after transfection of CV-1 cells. Finally, transient expression in 293 cells of cDNAs encoding the family members NF1-A4, NF1-C2, and NF1-X2 specifically repressed transcription from the BKV late promoter approximately 3-, 10-, and 10-fold, respectively, in a DNA binding-dependent manner. We conclude that some members of the NF1 family of transcription factors can act as sequence-specific cellular repressors of the BKV-MLP. We propose that titration of these and other cellular repressors by viral genome amplification may be responsible in part for the replication-dependent component of the early-to-late switch in BKV gene expression.

Animals↗
PubMed2001

Identification of a novel element involved in regulation of the lytic switch BZLF1 gene promoter of Epstein-Barr virus.

Epstein-Barr virus (EBV) is a human herpesvirus capable of establishing a latent state in B lymphocytes. EBV's BZLF1 gene product plays a central role in regulating the switch from latency to productive infection. Here, we identify a sequence element, 5'-CAGGTA-3', called ZV, located at nucleotides -17 to -12 relative to the transcription initiation site of the BZLF1 promoter. ZV sequence-specifically binds a cellular nuclear factor(s), ZVR. ZVR DNA-binding activity was present in the EBV-negative B-lymphocytic cell line DG75, the EBV-positive B-lymphocytic cell lines GG68 and 721, the cervical cell line C33A, and the kidney cell line CV-1 but not in the breast carcinoma cell line MCF-7. Mutations in ZV that relieve binding of ZVR lead to a two- to fourfold increase in basal expression of the BZLF1 promoter in DG75, C33A, and CV-1 cells. The same mutants exhibited a 40- to 180-fold increase in tetradecanoyl phorbol acetate-ionomycin-induced expression in DG75 cells and a 22-fold increase in C33A cells. Thus, ZVR functions as a regulator of the BZLF1 promoter, repressing transcription when bound to the ZV site in the absence of inducers. No differences in basal or induced transcription between wild-type and ZV mutant BZLF1 promoters were observed in ZVR-negative MCF-7 cells. ZVR failed to bind any of the previously identified negative regulatory elements within the BZLF1 promoter. We conclude that ZV functions as an important regulatory element of the BZLF1 promoter, with ZVR likely playing important roles in the maintenance of latency and reactivation of EBV.

Animals↗
PubMed2001

Critical roles of nuclear receptor response elements in replication of hepatitis B virus.

Functional analysis of the roles of the nuclear receptor response elements (NRREs) in the transcription and replication of hepatitis B virus (HBV) in the context of its whole genome has been hampered by the extensive overlapping of the NRREs with the regions encoding viral proteins. We introduced point mutations that inactivate the NRREs individually without altering the open reading frames of viral proteins. These mutations in the context of a plasmid containing 1.2 copies of the HBV genome were transiently transfected into the human hepatoma cell line Huh7. Inactivation of the NRRE in either the preC promoter (NRRE(preC)) or enhancer I (NRRE(enhI)) led to moderate reductions in synthesis of viral RNAs. Concurrent inactivation of both NRREs led to 7- to 8-fold reductions in synthesis of the preC, pregenomic, and preS RNAs and a 15-fold reduction in synthesis of the S RNA. The accumulation of viral DNA in the cytoplasmic nucleocapsids and virion particles in the culture medium was also reduced seven- to eightfold. These results suggest that these NRREs are critical for the efficient propagation of HBV in hepatocytes. In cotransfection experiments we also found that overexpression of PPARalpha-RXRalpha in the presence of their respective ligands led to a fourfold increase in pregenomic RNA synthesis and a four- to fivefold increase in viral DNA synthesis, while it had little or no effect on synthesis of the other viral RNAs. Similar effects were observed with overexpression of PPARgamma-RXRalpha in the presence of their respective ligands. This activation was dependent on NRRE(preC), because the increase in synthesis of viral RNA and DNA was not observed when this site was mutated. Likewise, no activation of synthesis of pregenomic RNA and viral DNA by PPARalpha-RXRalpha was observed in a naturally occurring NRRE(preC)(-) mutant of HBV. Our results suggest that interactions between nuclear receptors and NRREs present in the HBV genome may play critical roles in regulating its transcription and replication during HBV infection of hepatocytes.

Base Sequence↗
PubMed1997

The prediction of the structure of technician (V) complexes using density-functional techniques.

Electronic structure calculations using the local-density functional method were shown to accurately reproduce the geometry of five oxo-Tc (V)-based radiopharmaceuticals containing a variety of donor ligands. Calculations were performed using the DGauss program on a Cray C90 supercomputer, requiring approximately 40 CPU h for each molecule. The predicted bond lengths were within +/- 0.04 A of the crystallographic coordinates, and bond angles within +/- 4 degrees. Root mean square (RMS) deviations between the predicted and crystallographic coordinates were less than 0.06, and visual inspection demonstrated nearly perfectly superimpossible structures.

Organotechnetium Compounds↗
PubMed1997

Direct modulation of simian virus 40 late gene expression by thyroid hormone and its receptor.

Transcription of the late genes of simian virus 40 (SV40) is repressed during the early phase of the lytic cycle of infection of primate cells by the binding of cellular factors, called IBP-s, to the SV40 late promoter; repression is relieved after the onset of viral DNA replication by titration of these repressors (S. R. Wiley, R. J. Kraus, F. R. Zuo, E. E. Murray, K. Loritz, and J. E. Mertz, Genes Dev. 7:2206-2219, 1993). Recently, we showed that IBP-s consists of several members of the steroid/thyroid hormone receptor superfamily (F. Zuo and J. E. Mertz, Proc. Natl. Acad. Sci. USA 92:8586-8590, 1995). Here, we show that the thyroid hormone receptor TRalpha1, in combination with retinoid X receptor alpha (RXRalpha), is specifically bound at the transcriptional initiation site of the major late promoter of SV40. This binding repressed transcription from the SV40 late promoter by preventing the formation of pre-initiation complexes. Addition of the thyroid hormone 3,5,3'-L-triiodothyronine (T3) resulted in reversal of this repression in cotransfected CV-1 cells. Interestingly, repression did not occur when this thyroid response element (TRE) was translocated to 50 bp upstream of the major late initiation site. Binding of TRalpha1/RXRalpha heterodimers to this TRE induced bending of the promoter DNA. We conclude that hormones and their receptors can directly affect the expression of SV40, probably by affecting protein-protein and protein-DNA interactions involved in the formation of functional preinitiation complexes.

Animals↗
PubMed1997

Differential regulation of the pre-C and pregenomic promoters of human hepatitis B virus by members of the nuclear receptor superfamily.

Synthesis of the pre-C and pregenomic RNAs of human hepatitis B virus (HBV) is directed by two overlapping yet separate promoters (X. Yu and J. E. Mertz, J. Virol. 70:8719-8726, 1996). Previously, we reported the identification of a binding site for the nuclear receptor hepatocyte nuclear factor 4 (HNF4) spanning the TATA box-like sequence of the pre-C promoter. This HNF4-binding site consists of an imperfect direct repeat of the consensus half-site sequence 5'-AGGTCA-3' separated by one nucleotide; i.e., it is a DR1 hormone response element (HRE). We show here that other receptors, including chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1), human testicular receptor 2 (TR2), and peroxisome proliferator-activated receptors (PPARs) as heterodimers with retinoid X receptors (RXRs), can also specifically bind this DR1 HRE. Synthesis of the pre-C and pregenomic RNAs was affected both in transfected hepatoma cells and in a cell-free transcription system by the binding of factors to this DR1 HRE. Interestingly, whereas some members of the hormone receptor superfamily differentially repressed synthesis of the pre-C RNA (e.g., HNF4 and TR2) or activated synthesis of the pregenomic RNA (e.g., PPARgamma-RXRalpha), other members (e.g., COUP-TF1) coordinately repressed synthesis of both the pre-C and pregenomic RNAs. Thus, HBV likely regulates its expression and replication in part via this DR1 HRE. These findings indicate that appropriate ligands to nuclear receptors may be useful in the treatment of HBV infection.

Base Sequence↗
PubMed1997

Estrogen-related receptor alpha 1 functionally binds as a monomer to extended half-site sequences including ones contained within estrogen-response elements.

The human estrogen-related receptor alpha 1 (hERR alpha 1) is an orphan member of the steroid/thyroid hormone receptor superfamily. A cDNA encoding this protein was originally isolated on the basis of sequence similarity in its DNA-binding domain with estrogen receptor alpha (ER alpha). Previously, we reported the purification of hERR alpha 1 from HeLa cell nuclear extracts on the basis of its ability to bind two sites in the late promoter of simian virus 40 (SV40). We have now determined the primary structure and the DNA and protein binding specificities of hERR alpha 1 and developed in vivo and in vitro assays for its functional activities. hERR alpha 1 was found to bind as a monomer, with a high-affinity binding site containing the extended half-site sequence 5'-TCAAG-GTCA-3'. Binding sites for hERR alpha 1 were identified in many cellular promoters, including some that were previously shown to function as estrogen-response elements (EREs). hERR alpha 1 was shown to function as a sequence-specific repressor of the SV40 late promoter in both cell culture and cell-free transcription systems. It was also shown to interact with both ER alpha and the transcription factor TFIIB by direct protein-protein contacts. Thus, hERR alpha 1 may play a role in the response of some genes to estrogen via heterodimerization with ERs or competition with ERs for binding to EREs.

Base Sequence↗
PubMed1996

Sequence of the polypyrimidine tract of the 3'-terminal 3' splicing signal can affect intron-dependent pre-mRNA processing in vivo.

Most pre-mRNAs require an intron for efficient processing in higher eukaryotes. However, not all introns can provide this function. For example, transcripts synthesized from a variant of the human beta-globin gene lacking its second intervening sequence (IVS2), yet retaining its first intervening sequence (IVS1), exhibit multiple defects in mRNA biogenesis. To investigate why, we transfected into monkey cells plasmids containing the human beta-globin gene and variants of it altered in (i) IVS1, (ii) the 3'-terminal exon, and (iii) the polyadenylation signal. The beta-globin RNAs accumulated in these cells were analyzed by quantitative S1 nuclease mapping for nuclear accumulation, intron excision, polyadenylation and cytoplasmic accumulation. We found that the 3' splicing signal of IVS1, with multiple purines interrupting its polypyrimidine tract, could efficiently function as an internal 3' splicing signal; however, it could not efficiently function as the 3'-terminal 3' splicing signal for any of these steps in intron-dependent mRNA biogenesis unless (i) its polypyrimidine tract was made uninterrupted in pyrimidines, or (ii) specific sequences were deleted from the 3'-terminal exon. We conclude that whether an intron can provide the function necessary for efficient processing of intron-dependent pre-mRNA is dependent upon the ability of its 3' splicing signal to define the 3'-terminal exon. On the practical side, this finding means one needs to consider both the sequence and location of the intron to be included in an intron-dependent gene to obtain efficient expression in vivo.

Animals↗
PubMed1996

Experimentally determined weight matrix definitions of the initiator and TBP binding site elements of promoters.

The basal elements of class II promoters are: (i) a-30 region, recognized by TATA binding protein (TBP); (ii) an initiator (Inr) surrounding the start site for transcription; (iii) frequently a downstream (+10 to +35) element. To determine the sequences that specify an Inr, we performed a saturation mutagenesis of the Inr of the SV40 major late promoter (SV40-MLP). The transcriptional activity of each mutant was determined both in vivo and in vitro. An excellent correlation between transcriptional activity and closeness of fit to the optimal Inr sequence, 5'-CAG/TT-3', was found to exist both in vivo and in vitro. Employing a neural network technique we generated from these data a weight matrix definition of an Inr that can be used to predict the activity of a given sequence as an Inr. Using saturation mutagenesis data of TBP binding sites we likewise generated a weight matrix definition of the -30 region element. We conclude the following: (i) Inrs are defined by the nucleotides immediately surrounding the transcriptional start site; (ii) most, if not all, Inrs are recognized by the same general transcription factor(s). We propose that the mechanism of transcription initiation is fundamentally conserved, with the formation of pre-initiation complexes involving the concurrent binding of general transcription factors to the -30, Inr and, possibly, downstream elements of class II promoters.

Animals↗
PubMed1996

Promoters for synthesis of the pre-C and pregenomic mRNAs of human hepatitis B virus are genetically distinct and differentially regulated.

Two similar, yet functionally distinct genomic RNAs are transcribed from the DNA genome of the human hepatitis B virus. The pre-C RNAs encode the precore protein which is proteolytically processed to yield e antigen. The pregenomic RNAs encode both the nucleocapsid protein and reverse transcriptase and serve as the templates for viral DNA replication. To determine whether synthesis of these two RNAs is directed from a single or a closely spaced pair of promoters, we introduced point and insertion mutations into the basal elements of the promoter that directs their synthesis. Transcription from these mutants was examined both in cell-free transcription systems derived from hepatoma (HepG2) and nonliver (HeLa) cell lines and by transient transfection of hepatoma cell lines (Huh7 and HepG2). The data from these experiments indicated that synthesis of the pre-C and pregenomic RNAs is directed by two distinct promoters and that the basal elements of these two promoters partially overlap, yet are genetically separable, with each consisting of its own transcriptional initiator and a TATA box-like sequence situated approximately 25 to 30 bp upstream of its sites of initiation. A 15-bp insertion was found to be sufficient to physically separate these two promoters. Furthermore, these two promoters can be differentially regulated, with the transcriptional activator Sp1 specifically activating transcription from the pregenomic promoter and the hepatocyte nuclear factor 4 specifically repressing transcription from the pre-C promoter. Thus, we conclude that the promoters used in synthesis of the pre-C and pregenomic mRNAs are genetically distinct and separately regulated.

Base Sequence↗
PubMed1996

The major transcriptional transactivation domain of simian virus 40 large T antigen associates nonconcurrently with multiple components of the transcriptional preinitiation complex.

Simian virus 40 (SV40) large T antigen (Tag) is a promiscuous transcriptional transactivator; however, its mechanism of transactivation remains unknown. Recent studies have suggested the possible involvement of protein-protein interactions with TBP, the TATA box-binding protein of TFIID, and TEF-1, an enhancer-binding factor. We show here that (i) the Tag domain containing amino acids 133 to 249 directly interacts with the general transcription factor TFIIB, the activator protein Sp1, and the 140-kDa subunit of RNA polymerase II, as well as with TBP and TEF-1; (ii) these interactions can also occur when these transcription factors are present in their functional states in cellular extracts; (iii) binding of Tag to TBP is eliminated by preincubation of TBP either at 48 degrees C or with the adenovirus 13S E1a protein; (iv) this domain of Tag cannot bind concurrently to more than one of these transcription factors; and (v) the substitution of Tag amino acid residues 173 and 174 inactivates the ability of this Tag domain both to associate with any of these transcription factors and to transactivate the SV40 late promoter. Thus, we conclude that SV40 Tag probably does not transactivate via the concurrent interaction with multiple components of the preinitiation complex. Rather, we hypothesize that transactivation by Tag may primarily occur by removing or preventing the binding of factors that inhibit the formation of preinitiation complexes.

Antigens, Polyomavirus Transforming↗
PubMed1995

Simian virus 40 late gene expression is regulated by members of the steroid/thyroid hormone receptor superfamily.

Transcription of the late genes of simian virus 40 (SV40) is repressed during the early phase of the lytic cycle of infection of binding of cellular factors, called IBP-s, to the SV40 late promoter; repression is relieved after the onset of viral DNA replication by titration of these repressors. Preliminary data indicated that one of the major components of IBP-s was human estrogen-related receptor 1 (hERR1). We show here that several members of the steroid/thyroid hormone receptor superfamily, including testis receptor 2, thyroid receptor alpha 1 in combination with retinoid X receptor alpha, chicken ovalbumin upstream promoter transcription factors 1 and 2 (COUP-TF1 and COUP-TF2), as well as hERR1, possess the properties of IBP-s. These receptors bind specifically to hormone receptor binding sites present in the SV40 major late promoter. Recombinant COUP-TF1 specifically represses transcription from the SV40 major late promoter in a cell-free transcription system. Expression of COUP-TF1, COUP-TF2, or hERR1 in monkey cells results in repression of the SV40 late promoter, but not the early promoter, in the absence of the virally encoded large tumor antigen. Overexpression of COUP-TF1 leads to a delay in the early-to-late switch in SV40 gene expression during the lytic cycle of infection. Thus, members of this superfamily can play major direct roles in regulating expression of SV40. Possibly, natural or synthetic ligands to these receptors can serve as antiviral drugs. Our findings also provide the basis for the development of assays to screen for the ligands to testis receptor 2 and hERR1.

Antigens, Viral, Tumor↗
PubMed1995

HnRNP L binds a cis-acting RNA sequence element that enables intron-dependent gene expression.

Most pre-mRNAs require an intron for efficient processing in higher eukaryotes. To test the hypothesis that intron-independent gene expression involves positive, cis-acting RNA sequence elements, we constructed chimeric genes in which various regions of the naturally intronless HSV-TK gene were inserted into an intronless variant of the highly intron-dependent human beta-globin gene. Using a transient transfection assay, we identified a 119-nucleotide sequence element contained within the transcribed region of the HSV-TK gene that enables efficient cytoplasmic accumulation of globin RNA in the absence of splicing. RNA UV-cross-linking assays indicated that a 68-kD protein present in nuclear extracts of HeLa and COS cells specifically binds to this HSV-TK sequence element. This 68-kD protein was found to cross-react with an antiserum specific to hnRNP L. Recombinant hnRNP L was shown to bind with high sequence specificity to this RNA sequence element. Analysis of substitution mutants in this element indicated that binding of hnRNP L correlates with accumulation of the RNA in the cytoplasm. Thus, we conclude that (1) hnRNP L binds in a sequence-specific manner to this RNA sequence element that enables intron-independent gene expression, and (2) intron-independent pre-mRNA processing and transport involves sequence-specific RNA-protein interactions between cis-acting RNA sequence elements and proteins such as hnRNP L. This sequence element may be of general use for the efficient expression of cDNA versions of intron-dependent genes.

Animals↗
PubMed1993

Polyadenylation site selection cannot occur in vivo after excision of the 3'-terminal intron.

Splicing of 3'-terminal introns and polyadenylation of pre-mRNAs can be coupled in an appropriate cell-free system. However, definitive evidence has been lacking as to whether these events are coupled in vivo and whether the order of these two processing events is obligatory. Here, we investigated these questions by examining the in vivo processing of transcripts that differ solely by the precise insertion of an intron within the first of two polyadenylation signals. Quantitative S1 nuclease mapping and PCR techniques were utilized to analyze the processed RNAs that accumulated in monkey cells transfected with plasmids encoding these transcripts. We found that, whereas all of the primary transcripts that lacked the inserted intron were processed via utilization of the 5'-proximal polyadenylation signal, none of the transcripts initially disrupted in this signal were processed this way even though the disrupting intron had been properly excised and excision sometimes preceded polyadenylation. In addition, deletion of the second polyadenylation signal resulted in failure of spliced transcripts to accumulate. We conclude that selection of, but not necessarily cleavage at the polyadenylation site precedes excision of the 3'-terminal intron in vivo; although coupling exists during selection of the sites to be used for polyadenylation and excision of the 3'-terminal intron, the actual order of the subsequent enzymatic reactions is probably simply a reflection of their relative kinetics.

Animals↗
PubMed1993

On achieving better than 1-A accuracy in a simulation of a large protein: Streptomyces griseus protease A.

Computational methods are frequently used to simulate the properties of proteins. In these studies accuracy is clearly important, and the improvement of accuracy of protein simulation methodology is one of the major challenges in the application of theoretical methods, such as molecular dynamics, to structural studies of biological molecules. Much effort is being devoted to such improvements. Here, we present an analysis of a 187-ps molecular dynamics simulation of the serine protease Streptomyces griseus protease A in its crystal environment. The reproduction of the experimental structure is considerably better than has been achieved in earlier simulations--the root mean square deviation of the simulated structure from the x-ray structure being less than 1 A, a significant step toward the goal of simulating proteins to within experimental error. The use of a longer cutoff with truncation rather than a switching function, inclusion of all crystalline water and the counterions in the crystallization medium, and use of the consistent valence force field characterize the differences in this calculation.

Bacterial Proteins↗
PubMed1993

SV40 early-to-late switch involves titration of cellular transcriptional repressors.

We have purified factors from HeLa cell nuclear extracts that bind to the transcriptional initiation site of the SV40 major late promoter (SV40-MLP). The resulting fraction consists predominantly of three proteins, collectively called initiator-binding protein of SV40 (IBP-s) with electrophoretic mobilities of approximately 45-55 kD. Gel mobility-shift and DNase I-protection analyses indicate that each of these three proteins associates with high affinity to sequences located at the initiation site and 55 bp downstream of it. IBP-s-binding sites with lower affinities are located at +5 and +30. Addition of purified IBP-s to a cell-free transcription system represses transcription from the SV40-MLP, but not the SV40 early promoter. SV40 mutants lacking the two strongest IBP-s-binding sites (1) are not repressed by the addition of IBP-s in vitro, (2) overproduce late RNA (relative to wild-type SV40) at low, but not high, template copy number in vitro, and (3) exhibit increased levels of late RNA at early, but not late, times after transfection into CV-1 cells. Therefore, IBP-s is a cellular repressor of transcription of the SV40-MLP that may, in large part, be responsible for the replication-dependent component of the early-to-late shift in SV40 gene expression. Partial amino acid sequence data obtained from the approximately 55-kD component of IBP-s indicate that it is hERR1, an orphan member of the steroid-thyroid hormone receptor superfamily. These findings suggest simple molecular mechanisms by which hormones may modulate expression of viral late genes. We speculate that activation of expression of the late genes of other viruses may occur by similar mechanisms.

Animals↗