Biomedical subjects

K Khalili

Publications and source records attributed to K Khalili.

At least 163 records · Page 9Linked to original sources

PubMed1991

Regulation of JCVL promoter function: evidence that a pentanucleotide "silencer" repeat sequence AGGGAAGGGA down-regulates transcription of the JC virus late promoter.

The human neurotropic papovavirus JCV contains sequences within the two 98-bp tandem repeats which play a key role in glial-specific transcription of the viral early and late promoters. Previous analysis of the 98-bp sequence has delineated several protein-binding domains that are recognized by nuclear factors present in human brain cells. In the present study, by deletion mutation analysis, we have identified a region within each 98-bp repeat that reduces transcriptional activity of the JCV late promoter (JCVL). Using synthetic oligonucleotides spanning this region, designated "OP," we demonstrate that down-regulation of the JCVL promoter is associated with a pentanucleotide repeat sequence (AGGGAAGGGA) juxtaposed to the poly(dA) tract within the 98-bp tandem repeats. The OP sequence interacts specifically with a protein derived from glial nuclear extract and forms a major 56- to 60-kDa complex. Methylation interference experiment indicates that the three G residues proximal to the poly(dA) tract make major groove contacts with the protein. Single-base-pair substitution of these residues suggests that the complex can form in the presence of two of the three guanosyl residues. The possible role of this protein in regulating the JCV lytic cycle in concert with nearby regulatory elements within JCV promoter region is discussed.

Animals↗

PubMed1990

Regulation of a human neurotropic virus promoter, JCVE: identification of a novel activator domain located upstream from the 98 bp enhancer promoter region.

Transcription of the human neurotropic virus promoter, JCVE, and its regulation in glial cells are controlled by the 98 bp tandem repeats positioned between the viral early and late genes. Here, we show that a region, designated domain-D, located upstream from the 98 bp repeats functions as a transcriptional activator and increases JCVE promoter activity. Using the reporter SV40E promoter fused to the bacterial chloramphenicol acetyltransferase (CAT) gene, we demonstrate that domain-D stimulates the basal SV40E promoter activity in glial and to a lesser degree in HeLa cells. Results from gel mobility-shift assays indicate that domain-D interacts with proteins derived from glial and HeLa extracts and results in the formation of specific DNA-protein complexes. Through UV cross-linking assays, we demonstrate that these complexes have similar electrophoretic mobilities which comigrate with the 43-50 Kd proteins derived from glial and HeLa cells. These findings, together with our previous observations, imply that the JCVE control region is composed of multiple common and specific activator domains that may account for the increased expression of the promoter in glial cells. The possible role of the D-binding protein in transcription of the JCVE promoter is discussed.

Animals↗

PubMed1990

Myelin basic protein gene transcription. Identification of proximal and distal cis-acting regulatory elements.

Myelin basic proteins (MBPs) represent a major component of the myelin membrane which are exclusively expressed by glial cells in the nervous system. The cell type-specific expression of MBP is controlled preferentially at the level of RNA synthesis. To investigate the mechanisms by which the MBP gene is regulated, we analyzed transcriptional regulation of this gene in glial and non-glial cells. We have demonstrated that the 320 base pairs upstream of the MBP transcriptional start site contain regulatory elements that preferentially stimulate transcription of MBPs in glial cells. Using a test vector containing the simian virus 40 (SV40) early promoter placed upstream of the bacterial chloramphenicol acetyltransferase gene, we localized three major promoter elements within the 5'-upstream sequence. These elements, designated MB1, MB4, and MB7, spanning proximal (-14 to -50) and distal (-130 to -169 and -249 to -288) positions with respect to the RNA initiation site, activated SV40 promoter transcription more than 40-fold in glial cells. The promoter distal elements, MB4 and MB7, enhanced SV40 promoter activity 2- and 8-fold, respectively, in L cells. Using the gel mobility shift assay, we have demonstrated that the MBP activators (MB1, MB4, and MB7) interact with multiple proteins derived from glial and L cell extract and result in the formation of several complexes. Comparison of band intensity of these complexes implies that these cells contain both unique and ubiquitous DNA binding proteins that recognize the DNA sequences within these activators. These studies suggest that the MBP promoter consists of several regulatory sequences in which the proximal element, MB1, and one of the distal elements, MB4, are selectively more active in glial cells than in L cells. Thus, these novel regulatory elements, in concert with other sequences, appear to stimulate MBP promoter transcription in glial cells.

Animals↗

PubMed1990

A nuclear protein derived from brain cells stimulates transcription of the human neurotropic virus promoter, JCVE, in vitro.

The 98-base pair enhancer/promoter sequence is critical for cell type-specific transcription of the human neurotropic viral promoter, JCVE, in glial cells. Transcriptionally active extracts were prepared from glial cells and used to identify cis- and trans-acting regulatory elements that are involved in the glial-specific activation of the JCVE promoter. Results indicate that multiple regulatory sequences within the 98-base pair repeat specifically bind to nuclear proteins present in glial cells and positively regulate viral early RNA synthesis. The central region of the repeat, designated domain-B, interacts with a 45-kDa nuclear protein present in brain cells. This brain-specific protein was purified by conventional and DNA affinity chromatography. Complementation of the highly purified protein with HeLa extract significantly increased JCVE promoter activity. Thus, association of the novel glial-origin transcription factor with its target sequence increases transcription of the JCVE promoter in a non-glial context.

Animals↗

PubMed1990

Trans-activation of the JC virus late promoter by the tat protein of type 1 human immunodeficiency virus in glial cells.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by the JC virus (JCV), a human papovavirus. PML is a relatively rare disease seen predominantly in immunocompromised individuals and is a frequent complication observed in AIDS patients. The significantly higher incidence of PML in AIDS patients than in other immunosuppressive disorders has suggested that the presence of human immunodeficiency virus type 1 (HIV-1) in the brain may directly or indirectly contribute to the pathogenesis of this disease. In the present study we have examined the expression of the JCV genome in both glial and non-glial cells in the presence of HIV-1 regulatory proteins. We find that the HIV-1-encoded trans-regulatory protein tat increases the basal activity of the JCV late promoter, JCVL, in glial cells. In a reciprocal experiment, the JCV early protein, the large tumor antigen, stimulates expression from JCVL and HIV-1 long terminal repeat promoter in both glial and non-glial cells. This trans-activation occurs at the level of RNA synthesis, as measured by the rate of transcription, stability of the message, and translation. We conclude that the presence of the HIV-1-encoded tat protein may positively affect the JCV lytic cycle in glial cells by stimulating JCV gene expression. Our results suggest a mechanism for the relatively high incidence of PML in AIDS patients than in other immunosuppressive disorders. Furthermore, our findings indicate that the HIV-1 regulatory protein tat may stimulate other viral and perhaps cellular promoters, in addition to its own.

Cell Line↗

PubMed1990

Double-stranded RNA unwinding and modifying activity is detected ubiquitously in primary tissues and cell lines.

A double-stranded RNA unwinding and modifying activity was found to be present in a wide range of tissues and cell types. The level of activity did not vary significantly with respect to the state of cell differentiation, cell cycle, or transformation. Thus, the unwinding and modifying activity, localized in the nucleus in somatic cells and capable of converting many adenosine residues to inosine, appears to be one of the housekeeping genes.

Animals↗

PubMed1990

Nuclear proteins in mouse brain cells bind specifically to the myelin basic protein regulatory region.

Expression of myelin basic protein (MBP) in mice is regulated in a cell- and stage-specific manner during brain development. The MBP control region contains multiple cis-acting elements, shown by in vivo and in vitro assays, which are responsible for its unique pattern of transcription. Using synthetic DNA fragments spanning the MBP control region, we have analyzed nuclear proteins obtained from newborn (2-3 d), young adult (18-30 d), and adult (60 d) animals; these nuclear proteins form DNA-protein complexes with the MBP regulatory region. Brain extracts from young adult and adult mice showed enhanced binding activities with the sequences supporting transcriptional activation in glial cells. Deletion analysis of the proximal activating sequence located at position -14 to -50 with respect to the RNA initiation site resulted in identification of a small region, located between nucleotides -14 to -37, which is required for formation of the complexes. Southwestern assay revealed a major 39-kD protein from young adult brain extract that recognizes the sequences between nucleotides -14 to -37. An additional minor 37-kD protein, derived from young adult brain extract, was also found to be associated with this proximal activating region. Of particular interest is the observation that the minor 37-kD protein became more abundant in the extract derived from adult brain, whereas the major 39-kD protein became less abundant. The possible role of these proteins in cell/stage-specific transcription of MBP is discussed.

Animals↗

PubMed1990

Regulation of the human neurotropic virus promoter by JCV-T antigen and HIV-1 tat protein.

We compared the ability of HIV-1 tat protein and JCV T-antigen in inducing transcription from the JCV late promoter, JCVL. A JCVL promoter-chloramphenicol acetyltransferase plasmid (pJCL-CAT) was transfected into human glial cells alone or together with plasmids producing T-antigen and tat protein. CAT enzyme activity obtained from the transfected cells indicated that both JCV T-antigen and HIV-1 tat proteins stimulated JCV late gene expression. However, the level of induction mediated by tat protein was significantly higher than that obtained with T-antigen. Moreover, in contrast to JCV T-antigen, tat stimulated JCVL-promoter activity over a narrow range of ptat expressor plasmid concentration. Co-transfection of both T-antigen and tat plasmids at optimal concentrations resulted in greater than additive CAT activity from the JCVL promoter. This synergism suggests that the two activator proteins utilize alternative mechanisms to exert their effects. Using deletion mutations from the 5' end of the JCVL promoter, we demonstrated that different regions within the JCV enhancer/promoter are important for T-antigen and tat induction, implying that these activators function through distinct targets to increase JCVL promoter activity.

Antigens, Polyomavirus Transforming↗

PubMed1989

Regulation of JCVL promoter function: transactivation of JCVL promoter by JCV and SV40 early proteins.

To better understand the basis of cell type specificity of JCV replication, we have analyzed the expression of the viral late promoter in glial cells. Using transient transfection procedures, we show that the late gene expression, like that of the early gene, is restricted to glial cells. However, cotransfection with a plasmid producing the JCV early protein, T-antigen, stimulates expression from the JCV late promoter in both glial and non-glial cells. The SV40-encoded T-antigen acts similarly on transcription of JCV late promoter in both cell types. This transacting effect occurs at the level of RNA synthesis, as measured by the rate of transcription, stability of the message, and translation. These results indicate that basal JCV late promoter activity is restricted to glial cells, whereas in the presence of viral early protein this promoter functions in both glial and non-glial cells.

Antigens, Polyomavirus Transforming↗

PubMed1989

Cell type-specific expression of JC virus early promoter is determined by positive and negative regulation.

We analyzed control sequences of the human papovavirus JC virus (JCV) to define the cis-acting elements that regulate specific expression of the viral early region genes in glial cells. Nuclear run-on transcription, S1 analysis, and chloramphenicol acetyltransferase enzyme activity in a transient transfection assay established that the cell type-specific expression of JCV early genes is determined at the transcriptional level. Using DNase footprinting analysis of nuclear proteins prepared from glial and nonglial cells, we located four regions within the JCV control sequences that specifically interacted with the proteins. In glial cells, all four domains contributed to the specific expression of a heterologous promoter, whereas in nonglial cells, two protein-binding regions showed no effect on basal transcriptional activity and the other two domains significantly downregulated transcription of the promoter. We conclude that cell type-specific transcription of the JCV early promoter is under both positive and negative regulation in eucaryotic cells.

Base Sequence↗

PubMed1989

The acidic amino-terminal region of the HIV-1 Tat protein constitutes an essential activating domain.

The Tat protein encoded by the human immunodeficiency virus (HIV) is an efficient activator of HIV gene expression. Many eukaryotic transcriptional activators contain a nucleic acid binding domain and a separate activating domain. These activating regions are acidic and often amphipathic. The amino terminus of the HIV-1 Tat protein is acidic with a periodicity of acidic, polar, and hydrophobic residues consistent with that of an amphipathic alpha helix. This region appears to be important for Tat function. We have analyzed the functional significance of acidic residues within the amino-terminal region of Tat by means of site-directed mutagenesis and by testing the capacity of mutant proteins to trans-activate the viral long terminal repeat (LTR) Conservative changes (acidic to acidic) were well tolerated, whereas acidic to neutral and acidic to basic changes markedly reduced Tat activity. The relative importance of each of the three acidic residues correlated with proximity to the amino terminus. Substitution of the entire domain with heterologous sequences that might form an acidic, amphipathic alpha helix partially restored activity when compared with an amino-terminal truncation mutant. In contrast to the observed importance of acidic residues, hydroxylated residues between amino acids 40 and 47 were dispensable for Tat function. These data suggest that the acidity of the amino terminal region is important for Tat function and that Tat-mediated trans-activation may be similar to that of other known activator proteins.

Amino Acid Sequence↗

PubMed1988

Nuclear factors in human brain cells bind specifically to the JCV regulatory region.

The human polyomavirus, JCV, differs from other papovaviruses in its tissue tropism for human glial cells. Transcription of the early region of the virus, at least in part, contributes to the tissue specificity of JCV. In this study, we have synthesized oligonucleotides which span the JCV 98 bp repeat unit. Using gel mobility shift and UV cross-linking assays, we have demonstrated that four proteins from a human fetal brain extract interact specifically with the JCV promoter/enhancer. Two proteins of 82 kd and 78/80 kd recognize the 5'- and 3'-terminal regions of the JCV 98 bp repeat sequence, respectively. The mol. wt of these proteins are similar in HeLa and brain extracts. In contrast, the proteins which recognize the central region of the 98 bp enhancer are distinct in HeLa (85 kd) and fetal brain (45 kd) extracts. The possible role of these proteins in tissue-specific expression of the JCV early promoter in brain cells is discussed.

Base Sequence↗

PubMed1988

Carboxyl-terminal mutants of the large tumor antigen of simian virus 40: a role for the early protein late in the lytic cycle.

Simian virus 40 (SV40) mutants dl1066 and dl1140 contain deletions within the region encoding the carboxyl terminus of the large tumor (T) antigen. Although these mutations have little effect on the efficiency of viral DNA replication, they decrease the yield of infectious virus particles by 3-4 orders of magnitude [Pipas, J. (1985) J. Virol. 54, 569-575]. Here we show that the level of late RNA is lower by a factor of 5-15 in CV-1P monkey cells infected with these mutants compared to cells infected with wild-type SV40. Consistent with this decrease in RNA, synthesis of late viral structural proteins VP1 and VP3 decreases by a factor of 5-15. In contrast, the synthesis of SV40 agnoprotein decreases by a factor greater than 100. Intercistronic complementation of these mutants with pm1493 and dl121, two SV40 mutants that are defective in agnoprotein but encode wild-type T antigen, results in an increased synthesis of agnoprotein in the infected cells. These results suggest that the carboxyl-terminal portion of T antigen participates in the posttranscriptional regulation of agnoprotein.

Animals↗

PubMed1987

Translational regulation of SV40 early mRNA defines a new viral protein.

SP6-initiated in vitro transcripts, representing the three major classes of early SV40 mRNAs, early-early (EE) and two late-early (LE) transcripts, were assayed by in vitro translation to compare their relative efficiencies for synthesis of the SV40 T antigens. The presence of one or two potential AUG initiator codons in the leader sequences of the LE RNAs inhibits efficient translation from the downstream T-antigen initiator AUG. In vitro translation of the capped form of the shorter SV40 LE RNA resulted in the synthesis of a 2.7-kd protein. In vivo pulse labeling of SV40-infected CV-1 cells demonstrated the accumulation of a peptide of similar size at late times after lytic infection, indicating that it is an authentic viral protein encoded by the early leader sequence of SV40.

Animals↗

PubMed1987

Evidence for a shift in 5'-termini of early viral RNA during the lytic cycle of JC virus.

We have used primer extension and S1 analysis to localize the 5'-termini of JC virus (JCV) early RNAs in infected primary human fetal glial cells at various times postinfection and in stable JCV-transformed hamster fetal glial cells. At early times postinfection (Days 1-5), two early transcripts are initiated at nucleotides 5122 and 5082. A major shift in 5'-ends at later times results in the synthesis of a new series of early mRNAs beginning upstream at nucleotide 35 and downstream at nucleotides 5047, 5037, and 5012. In the transformed hamster cells, however, only one RNA species was detected, starting at nucleotide 5122. The mechanism underlying the shift in the initiation site of JCV early RNAs during a lytic infection remains unclear but appears analogous to that which occurs in the SV40 lytic cycle. Since the shift occurs during DNA replication, when T-antigen is at maximal levels, it is possible that T-antigen binding to JCV DNA and/or alterations in chromatin structure contribute to this event.

Animals↗

PubMed1987

Regulation of the host range of human papovavirus JCV.

Human papovavirus JCV is associated with the human demyelinating disorder progressive multifocal leukoencephalopathy. In tissue culture, the virus is largely restricted to growth in primary human fetal glial cell. In this study, we demonstrate two levels of regulation of the viral host range. Expression of the early JCV mRNA, which encodes the essential viral protein, large tumor antigen (T antigen), depends on recognition of the early enhancer/promoter elements by tissue-specific factors found in both human and rodent glial cells. In the presence of JCV T antigen, viral DNA replication requires a species-specific factor, presumably a component of DNA polymerase, which is found in a wide range of primate cells. We further demonstrate that simian virus 40 T antigen has sufficient homology to efficiently substitute for the analogous JCV protein in initiating viral DNA replication.

Animals↗

PubMed1986

Spacing between simian virus 40 early transcriptional control sequences is important for regulation of early RNA synthesis and gene expression.

We have analyzed the effect of insertion mutants between the simian virus 40 (SV40) 21-base pair (bp) repeats and the early-early (EE) TATA sequence. Insertion of 4, 42, or 90 bp of DNA at the SV40 NcoI site (map position 37) has been analyzed for its effect on expression of the SV40 early gene and positioning of the RNA 5' ends. Insertion of 4 bp reduced SV40 early promoter-dependent chloramphenicol acetyltransferase (CAT) expression by six- to eightfold. Increasing the size of the insertion to 42 or 90 bp resulted in a further drop in early gene expression to basal levels. At the RNA level, the 4-bp insertion reduced EE RNA synthesis approximately 10-fold. No concomitant increase in late-early (LE) RNA synthesis was observed. Insertion of 42 or 90 bp of DNA resulted in a decrease of EE RNA synthesis and a stimulation of LE RNA synthesis. Deletion of the SV40 72-bp repeats from the insertion mutants demonstrated that some, but not all, of the LE RNA depends upon the presence of these sequences. These studies suggest that the ability of RNA polymerase II to utilize the EE (TATA-directed) transcriptional control sequence requires an interaction with the upstream 21-bp repeats or the 72-bp repeats or both. That LE RNA levels in pJI1-in42 CAT and pJI1-in90 CAT were equivalent to the level of EE RNA in pJI1-CAT, yet the level of CAT gene expression was decreased greater than 10-fold, suggests that LE mRNA is under translational control and probably prefers a 5' initiation codon proximal to that of the CAT gene.

Base Sequence↗

PubMed1985

The gene encoding the large subunit of human RNA polymerase II.

As a first step to approach the structural and functional analysis of DNA-dependent RNA polymerase II (EC 2.7.7.8), we have isolated genomic sequences for the large subunit of the human enzyme. The sequences homologous to Drosophila RNA polymerase II large subunit sequences are present in the genome as single copy genes, when assayed at high stringency. The polypeptide information is encoded in a mRNA of 7.35 kilobases, as determined by Northern blot analysis. In vitro translation reveals a polypeptide of 220 kDa, similar in electrophoretic mobility to the largest subunit of the enzyme. A fusion-polypeptide synthesized in bacteria contains a region that cross-reacts with anti-RNA polymerase II antiserum. Antiserum directed against the purified fusion protein reacts with the large subunit of RNA polymerase II, whether in the intact IIA (220 kDa) or in the degraded IIB (180 kDa) forms. Moreover, the antifusion protein antibody inhibits not only the purified calf thymus RNA polymerase II activity but also specific RNA polymerase II transcription in a HeLa cell extract. Thus, the DNA fragment isolated contains structural and functional domains of the human RNA polymerase II large subunit.

Cell Line↗