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B M Peterlin

Publications and source records attributed to B M Peterlin.

At least 91 records · Page 5Linked to original sources
PubMed1990

Trans-activation by HIV-1 Tat via a heterologous RNA binding protein.

The HIV-1 trans-activator Tat increases levels of viral gene expression and replication. The target for Tat is an RNA stem-loop called TAR, located at the 5' end of all viral transcripts. To study the mechanism of action and map functional domains of Tat, we fused Tat to the coat protein of bacteriophage MS2, an RNA binding protein. TAR in the HIV-1 LTR was replaced by the operator, the RNA target of the coat protein. The hybrid Tat-coat protein trans-activated HIV-1 LTRs containing either TAR or operator sequences. Mutations in the operator that weaken binding of the coat protein in vitro led to decreased levels of trans-activation in vivo. Deletions in Tat within the hybrid Tat-coat protein identified activation and RNA binding domains of Tat. These experiments suggest that trans-activation by Tat can occur independently of TAR RNA and DNA binding proteins and that Tat exerts its effects on HIV-1 transcription by directly interacting with the TAR RNA stem-loop.

Amino Acid Sequence↗
PubMed1990

Major histocompatibility complex genes and susceptibility to systemic lupus erythematosus.

Susceptibility to systemic lupus erythematosus is associated with major histocompatibility complex (MHC)--encoded genes. We have used nucleotide sequence analysis to better define the disease-associated MHC alleles. HLA-DR2, DQw1, and especially the rare allele DQ beta 1. AZH confer high relative risk (RR = 14) for lupus nephritis in a Caucasian population of patients. Pilot studies using historical controls suggest that these genes also confer a high risk in non-Caucasian ethnic groups (RR = 24-78). We have found that DR4 is significantly decreased in patients with lupus nephritis. Fifty percent of the patients with lupus nephritis had either the DQ beta 1.1, the DQ beta 1.AZH, or the DQ beta 1.9 alleles. These alleles share amino acid residues that have been predicted to be the contact points for antigen and the T cell receptor. These HLA alleles appear to have a direct role in the predisposition to lupus nephritis, whereas DR4 may have a "protective" effect.

Alleles↗
PubMed1990

Cytomegalovirus activates transcription directed by the long terminal repeat of human immunodeficiency virus type 1.

Proteins encoded by a variety of DNA viruses activate gene expression from the promoter within the long terminal repeat (LTR) of the human immunodeficiency virus type 1 (HIV-1). The mechanism by which immediate-early (IE) gene products of human cytomegalovirus (CMV) activate expression from the HIV-1 LTR was examined in transient expression assays in cultures of human cells by using plasmids containing the LTR linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and a plasmid expressing the CMV IE gene. Analysis of clustered site mutations within the HIV-1 LTR revealed that sequences from nucleotides -6 to +20 (relative to the start site of transcription) are critical for responsiveness to transactivation by CMV IE gene products. This region partially overlaps the trans-acting response element (+19 to +42) required for function of the HIV-1 transactivator. The CMV IE gene was shown to increase the steady-state levels of both prematurely terminated and full-length transcripts initiated within the LTR. These results support a model in which CMV IE gene products act through a specific regulatory element in the HIV-1 LTR to increase viral transcription.

Base Sequence↗
PubMed1990

Mutational analysis of the DRA promoter: cis-acting sequences and trans-acting factors.

Class II major histocompatibility genes are expressed at high levels in B lymphocytes and are gamma interferon (IFN-gamma) inducible in many other cells. Previously, we observed that DRA promoter sequences from positions -150 to +31 determine the tissue specificity of this class II gene. Moreover, Z and X boxes located between positions -145 and -87 conferred B-cell specificity and IFN-gamma inducibility upon a heterologous promoter. In this study, sequences from positions -145 to -35 in the DRA promoter were systematically mutated by using oligonucleotide cassettes. Z (-131 to -125), pyrimidine (-116 to -109), X (-108 to -95), Y (-73 to -61), and octamer (-52 to -45) boxes were required for B-cell specificity and, with the exception of the octamer box, for IFN-gamma inducibility. Z box and sequences flanking Z and X boxes helped to determine low levels of expression in T and uninduced cells. In phenotypically distinct cells, shared and distinct proteins bound to these conserved upstream sequences. However, few correlations between expression and DNA-binding proteins could be made. Similar proteins bound to Z and X boxes, and the Z box most likely represents a duplication of the X box.

Base Sequence↗
PubMed1989

Signaling through T lymphocyte surface proteins, TCR/CD3 and CD28, activates the HIV-1 long terminal repeat.

The state of T cell activation and proliferation controls HIV-1 replication and gene expression. Previously, we demonstrated that the administration of PHA and PMA to the human T cell line Jurkat activates the HIV-1 enhancer, which is composed of two nuclear factor kappa B (NF kappa B) binding sites. Here, we show that PMA alone is sufficient for this effect. In addition, activation of T cells through the surface proteins TCR/CD3 and CD28 increased gene expression directed by the HIV-1 long terminal repeat (LTR) to the same extent as PMA. Analysis of 5' deletions in the LTR revealed that the NF kappa B binding sites and sequences in the upstream U3 region are required for this response. Whereas cyclosporin A did not inhibit the effect of PMA, it reduced the effects of agonists to TCR/CD3 and CD28 on the LTR. H7, an inhibitor of protein kinase C (PKC), blocked the effects of all stimuli. Thus, PMA activates the NF kappa B sites through a PKC-dependent pathway while ligands to TCR/CD3 and CD28 activate the LTR through a cyclosporin A-sensitive, PKC-dependent pathway of T cell activation. We conclude that mechanisms involved in the expression of IL-2 and the alpha-chain of the IL-2R alpha genes also play a role in the regulation of HIV-1. Physiologic stimuli can activate HIV-1 gene expression; agents that block T cell activation also inhibit activation of the LTR. These observations might serve as a model for the regulation of HIV-1 gene expression in peripheral blood T cells.

Animals↗
PubMed1989

trans-activation by the hepatitis B virus X protein shows cell-type specificity.

The hepatitis B virus X protein can trans-activate transcription from many viral promoters. We show here that this trans-activation is cell-type but not species specific, and that only a subset of promoters is trans-activated in any particular cell type. Furthermore, the T/t-antigens from simian virus 40 suppress this trans-activation. We hypothesize that the X protein acts via cellular factors which are phylogenetically conserved and developmentally regulated and whose effects are blocked by the T/t-antigens.

Animals↗
PubMed1989

Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat.

The human immunodeficiency virus (HIV-1)-encoded trans-activator (tat) increases HIV gene expression and replication. Previously, we demonstrated that tat facilitates elongation of transcription through the HIV-1 long terminal repeat (LTR) and that short transcripts corresponding to prematurely terminated RNA are released and accumulate in the absence of tat. Here, using a transient expression assay, we tested clustered and compensatory mutations, as well as 3' deletions, in the trans-acting responsive region (tar) and observed that the primary sequence in the loop and secondary structure in the stem of the stem-loop in tar are required for trans-activation by tat. Insertions in the 5' region of tar revealed that tar must be near the site of HIV-1 initiation of transcription for trans-activation by tat. Deletions (3') and an insertion in tar demonstrated that an intact stem-loop is required for the recovery of prematurely terminated transcripts. Short and full-length transcripts were observed also with HIV type 2 (HIV-2) in the absence and presence of tat, respectively. We conclude that an intact stem-loop in tar is essential for trans-activation by tat and that initiation of transcription by HIV-1 promoter factors and elongation of transcription by tat are coupled.

Chloramphenicol O-Acetyltransferase↗
PubMed1988

Major histocompatibility complex associations with systemic lupus erythematosus.

This study focused on clinical subsets within systemic lupus erythematosus (SLE) in order to identify more homogeneous patient groups in which to define disease susceptibility gene(s). Analysis of the major histocompatibility complex gene products and genes with major histocompatibility complex class II and class III locus-specific probes and oligonucleotide probes for selected human leukocyte antigen DQ-beta alleles showed significant increases of human leukocyte antigen DR2 and the rare DQ-beta allele DR2-DQw1.AZH in the lupus nephritis patients compared with lupus patients without renal disease (relative risk = 8.3). C4A null was detected in one third of all of the SLE patients. In two thirds of the C4A null patients this was due to a DR3-associated C4A gene deletion. The remaining third may have a regulatory defect and this was DR2-associated. DR4 was significantly decreased in the nephritis patients in comparison with the non-renal SLE patients (relative risk = 0.3). A novel DQ-beta gene has been sequenced from two SLE patients that has not been observed in the normal population. Potential implications of these findings are discussed.

Complement C2↗
PubMed1988

Characterization of the human monocyte high affinity Fc receptor (hu FcRI).

The high affinity Fc receptor (FcRI) of a human monocytic cell line, U937, was further characterized using a previously described murine monoclonal antibody, FcRmAb32. This antibody immunoprecipitated a 70 K cell surface glycoprotein. A solid phase ligand binding assay and a solid phase immunoprecipitation assay were combined to confirm that the 70 K cell surface glycoprotein immunoprecipitated by FcRmAb32 is an IgG binding protein. N-glycanase digestion shows that at least 20% of the relative mobility of the 70 K FcRI glycoprotein is due to N-linked carbohydrate. FcRmAb32 immunoprecipitated a 70 K glycoprotein from biosynthetically labelled U937 cells that co-migrated with the surface iodinated glycoprotein on 2-dimensional gel electrophoresis. A 50 K protein, that is biosynthetically labelled but not accessible to surface iodination, which, bound to control antibodies was also present in FcRmAb32 immunoprecipitates. FcRmAb32 only bound the mature fully glycosylated form of FcRI. The 70 K FcRI was not phosphorylated constitutively nor when U937 cells were stimulated by PMA.

Antibodies, Monoclonal↗
PubMed1988

Trans-activation of the human immunodeficiency virus long terminal repeat by the hepatitis B virus X protein.

Human immunodeficiency virus 1 has been implicated as the main etiologic agent of the acquired immunodeficiency syndrome. However, other infectious agents may accelerate the progression of this disease. In particular, hepatitis B virus has been suggested as one such cofactor. Therefore, we have investigated the effects of hepatitis B virus gene products on expression of the human immunodeficiency virus I in transient transfection studies of Jurkat lymphoblastic T cells, using as reporter the chloramphenicol acetyltransferase gene coupled to the long terminal repeat of human immunodeficiency virus I. As measured by the amount of chloramphenicol acetyltransferase activity, gene expression directed by the human immunodeficiency virus I long terminal repeat increased approximately 10-fold in response to the hepatitis B virus X protein. This trans-activation by the X protein is multiplicative with the effect of phorbol esters and can be accounted for by an increase in the steady-state level of chloramphenicol acetyltransferase mRNA. Analysis of deletion and clustered point mutants in the long terminal repeat indicated that the X protein exerts its effect through multiple cis-acting sites. These results provide a possible molecular basis for the association of hepatitis B virus and the acquired immunodeficiency syndrome and confirm that the X protein is a transcriptional transactivator.

Acquired Immunodeficiency Syndrome↗
PubMed1988

B-cell-specific and interferon-gamma-inducible regulation of the HLA-DR alpha gene.

We investigated the cis-acting sequences that function in the B-cell-specific and interferon-gamma-inducible expression of the HLA-DR alpha gene, a human class II major histocompatibility complex gene. The effects of 5' deletions on the activity of the DR alpha promoter and the influence of upstream DR alpha promoter elements on the activity of the herpes simplex virus thymidine kinase promoter were examined by a transient transfection assay in human B-, T-, and fibroblast cell lines. We show that the DR alpha gene is regulated by positive and negative cis-acting sequences between positions -1300 and +31 from the site of initiation of transcription. We also demonstrate that the DR alpha promoter sequences from positions -116 to -92 and from -136 to -80 are the minimal sequences required for conferring B-cell specificity and interferon-gamma inducibility upon the Herpes simplex virus thymidine kinase promoter, respectively.

B-Lymphocytes↗
PubMed1988

Evidence for a trans-acting factor that regulates the transcription of class II major histocompatibility complex genes: genetic and functional analysis.

The study of specific trans-acting transcription factors in prokaryotes and lower eukaryotes has been greatly facilitated by genetic analysis of mutant strains deficient in such factors. We have developed such a system to study mammalian trans-acting factors that regulate the transcription of class II major histocompatibility complex genes, using the mutant cell lines RM2 and RM3. These cells, derived from the human B-cell line Raji, specifically fail to transcribe their class II major histocompatibility complex genes. Here we show that a transfected HLA-DR alpha class II major histocompatibility complex gene, like the endogenous HLA-DR alpha genes, is efficiently transcribed in Raji cells but not in RM2 or RM3 cells, demonstrating that the mutant cells are deficient in a specific trans-acting factor required for transcription of these genes. HLA-DR expression in RM2 and RM3 cells is rescued by fusion to another B-cell line but not by fusion to each other. Thus, the defects in the two cell lines are recessive and noncomplementing and define a locus whose wild-type product we designate TF-X1. We show that TF-X1 influences the activity of a 24-base-pair B-cell-specific cis-acting transcription element in the HLA-DR alpha promoter. However, in three different biochemical assays, we detect no difference between wild-type and mutant cells in the DNA-binding proteins that interact with these DNA sequences. Thus, the defective version of TF-X1 may be a DNA-binding protein that binds to the HLA-DR alpha promoter but fails to activate transcription. Alternatively, TF-X1 may not be a DNA-binding protein at all.

Base Sequence↗
PubMed1988

Transcription and replication of human immunodeficiency virus-1 in B lymphocytes in vitro.

We and others have shown that HIV-1 transcription and replication in vitro are increased by the binding of transcriptional enhancer DNA sequences in the HIV-1 long terminal repeat (LTR) to a cellular protein designated Nuclear Factor-kappa B (NF-kappa B), a trans-acting transcription factor which is present in activated but not in resting T cells. Because NF-kappa B is also expressed in resting B cells we suggested that B cells might provide an optimal intracellular environment for HIV-1 transcription. Using a transient transfection assay, we show that the HIV-1 LTR is indeed more efficiently transcribed in the B cell line Raji than in the T cell line Jurkat, and that this effect maps to the transcriptional enhancer (NF-kappa B binding site) of the LTR. Furthermore, we show that Raji cells which have been rendered CD4-positive by gene transfection are susceptible to HIV-1 infection, and that viral gene expression and replication are more efficient in these CD4-positive B cells than in CD4-positive T cells. These findings demonstrate the crucial role of the transcriptional enhancer in regulating HIV-1 expression and, since receptors for HIV-1 are expressed by some Epstein-Barr virus (EBV)-positive B cells, they also suggest that HIV-1 replication could occur in EBV-positive B cells in vivo.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Mutant human B cell lines deficient in class II major histocompatibility complex transcription.

By mutagenesis and selection, two independent Ia- mutant cell lines derived from the Ia+ human B cell line Raji were isolated. One mutant failed to express detectable surface HLA-DR and HLA-DQ molecules, while the other expressed very low levels of these molecules. mRNA encoding Ia molecules was correspondingly absent or reduced in these cell lines. In contrast, levels of class I major histocompatibility complex (MHC) mRNA and protein were normal in these cells. The class II MHC genes of the mutant cells were grossly intact, suggesting that the defective expression of these genes was due to an absent or abnormal trans-acting regulatory factor. In vitro nuclear run-on transcription assays and measurement of class II MHC mRNA stability suggested that the specific defect in class II MHC gene expression in both mutant cell lines was at the level of transcription initiation.

B-Lymphocytes↗
PubMed1987

A tissue-specific transcriptional enhancer is found in the body of the HLA-DR alpha gene.

We mapped cis-acting regulatory elements in the HLA-DR alpha gene, which encodes the monomorphic subunit of the HLA-DR heterodimer. Genomic fragments of HLA-DR alpha were placed 5' or 3' to the chloramphenicol acetyltransferase reporter gene, the transcription of which was initiated from the Herpes simplex thymidine kinase promoter. In transient expression assays, fragments from the body of the HLA-DR alpha gene were able to increase chloramphenicol acetyltransferase activity in a position-, orientation-, and promoter-independent yet tissue-specific fashion. These HLA-DR alpha cis-acting regulatory elements contain previously identified DNase I-hypersensitive sites and DNA sequences homologous to those found in other eukaryotic transcriptional enhancers.

Acetyltransferases↗
PubMed1987

Monoclonal antibody immunoprecipitation of cell membrane glycoproteins.

Procedural modifications facilitating the immunoprecipitation of cell surface-associated glycoproteins by monoclonal antibodies are presented. The use of complexes of antibodies coupled to protein A-Sepharose in place of antibodies directly coupled to Sepharose, and the inclusion of ATP and salt in the lysis buffer, is shown to markedly reduce the nonspecific binding of aggregated cytoskeletal proteins. These modifications result in low backgrounds while the specific membrane-associated proteins are still quantitatively immunoprecipitated.

Adenosine Triphosphate↗