Biomedical subjects

B K Felber

Publications and source records attributed to B K Felber.

71 records · Page 4Linked to original sources

PubMed1989

rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA.

rev (trs/art) is an essential human immunodeficiency virus type 1 (HIV-1) regulatory protein. rev increases the levels of the gag- and env-producing mRNAs via a cis-acting element in the env region of HIV-1, named rev-responsive element. Our results show that rev increases the stability of the unspliced viral mRNA, while it does not affect the stability of the multiply spliced viral mRNAs that do not contain the rev-responsive element. The study of mutated proviral constructs producing mRNA that cannot be spliced revealed that the effect of rev on stability is independent of splicing. Our experiments also indicate that rev promotes the transport of the viral mRNA containing the rev-responsive element from the nucleus to the cytoplasm. The proposed functions of rev are consistent with its nuclear localization as shown by immunofluorescence. The selective effects of rev on the levels of the viral mRNA suggest a model for feedback regulation by rev leading to a steady state of viral expression.

Cell Nucleus↗

PubMed1989

The rev (trs/art) protein of human immunodeficiency virus type 1 affects viral mRNA and protein expression via a cis-acting sequence in the env region.

The study of expression of several human immunodeficiency virus type 1 proviral mutants in human cells in the presence or absence of rev (trs/art) protein reveals that rev increases the levels of unspliced and env mRNA and the accumulated structural viral proteins. rev protein produced from appropriate expression vectors fully complements the rev-defective mutants. rev requires the presence of a specific cis-acting sequence for its function. This rev-responsive element sequence has been localized within a 520 base-pair fragment in the env region of human immunodeficiency virus type 1. gag and env expression is coordinately regulated by rev. Two independent cis-acting elements localized in the gag and env regions are responsible for the low levels of gag and env mRNA in the absence of rev. These elements are different than the rev-responsive element and act independent of each other.

DNA Mutational Analysis↗

PubMed1989

Cross-activation of the Rex proteins of HTLV-I and BLV and of the Rev protein of HIV-1 and nonreciprocal interactions with their RNA responsive elements.

The Rex regulatory proteins of human T-cell leukemia virus type I (HTLV-I) and bovine leukemia virus (BLV), and the Rev protein of human immunodeficiency virus type 1 (HIV-1), promote the cytoplasmic accumulation and translation of viral messenger mRNAs encoding structural proteins. Rev and Rex act through cis-acting elements on the viral RNA; these elements are named Rev- and Rex-responsive elements, or RRE and RXRE, respectively. We show that the Rex proteins of HTLV-I and BLV are interchangeable, but only the Rex protein of HTLV-I can substitute for Rev of HIV-1. Rex of HTLV-I and Rev of HIV-1 appear to act on RRE by similar mechanisms. Rev of HIV-1 does not act on the RXRE of HTLV-I or BLV. The nonreciprocal action of Rev and Rex suggests that these factors interact directly with the cis-acting RNA elements of the two viruses.

Animals↗

PubMed1988

A quantitative bioassay for HIV-1 based on trans-activation.

A bioassay that is based on trans-activation has been developed for the detection and quantitation of the human immunodeficiency virus type 1 (HIV-1). Indicator cell lines were constructed that contain the HIV-1 long terminal repeat ligated to the chloramphenicol acetyltransferase (CAT) gene. Infection of these cells by HIV activates the expression of CAT protein. Isolates of HIV-1 with divergent nucleotide sequences activated the indicator cell lines to a similar extent, approximately 500- to 1000-fold. Human T cell lymphotropic viruses types 1 and 2, equine infectious anemia virus, and herpes simplex virus 1 did not activate the indicator cell lines. Isolates of simian immunodeficiency virus and human T cell lymphotropic virus type 4 activated these cells to a much lesser extent, which suggests that these viruses contain similar, but distinct, trans-activators. This assay can be used for the detection, quantitation, and typing of HIV and for studying the effect of drugs on the replication of HIV in different cellular backgrounds.

Acetyltransferases↗

PubMed1987

The molecular basis of a familial apoE deficiency. An acceptor splice site mutation in the third intron of the deficient apoE gene.

The molecular basis of the familial apoE deficiency was investigated by gene cloning and comparative expression studies of the normal and the deficient apoE gene. For the latter studies the apoE genes were placed under the control of the mouse metallothionein I promoter in a bovine papilloma virus vector. The studies showed that in the normal gene the mouse metallothionein I promoter directs the synthesis of normal apoE mRNA and protein. In contrast, in the deficient apoE gene the same promoter directs the synthesis of two abnormal apoE mRNA forms, which are similar to those observed in the peripheral blood monocyte macrophage cultures of the patient. Restriction analysis of the cloned gene and partial DNA sequence has shown an A to G substitution in the penultimate 3' nucleotide of the third intron of the apoE gene. This single base substitution abolishes the correct 3' splice site thus creating two abnormally spliced mRNA forms. The smaller form contains 53 nucleotides and the larger form contains the entire third intron of the apoE gene. Both of these mRNA species contain chain termination codons within the intronic sequence and code for short apoE peptides that are not detectable by gel electrophoretic techniques. These findings show that this form of familial apoE deficiency results from a point mutation in the 3' splice junction of the third intron of the apoE gene. Furthermore, the expression system we have employed to study apoE deficiency can be utilized to analyze a broad spectrum of genetic diseases associated with mRNA processing mutations.

Animals↗

PubMed1986

Expression and characterization of the trans-activator of HTLV-III/LAV virus.

The human T-lymphotropic retrovirus HTLV-III/LAV encodes a trans-activator that increases viral gene expression. We expressed this trans-activator in animal cells and studied its structural and functional characteristics. The putative trans-activator protein was immunoprecipitated from overproducing stable cell lines and shown to migrate as a 14-kilodalton polypeptide on sodium dodecyl sulfate-polyacrylamide gels. S1 nuclease mapping experiments showed that the trans-activator increases the levels of steady-state messenger RNA transcribed from the viral long terminal repeat promoter. Sequences within the R region of the HTLV-III/LAV long terminal repeat are essential for trans-activation. Quantitations of messenger RNA and protein showed that the protein increase was greater than the messenger RNA increase in CV1 and HeLa cells, indicating that more than one mechanism was responsible for the trans-activation and that cell type-specific factors may determine the final level of trans-activation.

Cell Line↗

PubMed1986

Cis-acting sequences responsible for the transcriptional activation of human T-cell leukemia virus type I constitute a conditional enhancer.

Transcription from the long terminal repeat promoter of human T-cell leukemia virus type I is activated in the presence of a trans-activator protein, TA-I, encoded in the 3' part of the genome. A series of long terminal repeat mutants and hybrid promoter constructs have been studied in a transient expression assay for their ability to be activated in the presence of the trans-activator protein. The sequences responsible for trans-activation have properties similar to those of transcription enhancer elements. They act relatively independent of position and orientation and activate both the homologous as well as heterologous promoters only in the presence of the trans-activator protein. Therefore, the trans-activator protein of human T-cell leukemia virus type I acts via an inducible enhancement mechanism.

Base Sequence↗

PubMed1985

The pX protein of HTLV-I is a transcriptional activator of its long terminal repeats.

Expression of the pX protein of human T-cell leukemia virus type I (HTLV-I) in animal cells demonstrates that this protein is a specific transcriptional activator of the long terminal repeats (LTR) of HTLV-I. Several other promoters are not affected by pX. No lymphocyte-specific factors are required for this activation. pX can be detected in the nucleus of transfected monkey kidney cells (line CV1) by indirect immunofluorescence. These results indicate that the pX protein is essential for the replication cycle of the virus and that it may be directly involved in the immortalization of human lymphocytes by HTLV-I.

DNA, Recombinant↗

PubMed1984

Duplicated heavy metal control sequences of the mouse metallothionein-I gene.

We present evidence that two distinct regions of the DNA upstream from the mouse metallothionein-I gene contain metal-responsive regulatory sites. This result was obtained by analyzing a systematic series of deletion, insertion, duplication, and clustered point mutations introduced into cultured cells on a simian virus 40 plasmid vector. The two upstream regions contain a duplicated evolutionarily conserved DNA sequence. While either upstream region is sufficient to confer heavy metal responsiveness, both are required to give maximal levels of induced transcription.

Amino Acid Sequence↗

PubMed1983

In contrast to other Xenopus genes the estrogen-inducible vitellogenin genes are expressed when totally methylated.

The methylation-sensitive restriction enzymes Hha I and Hpa II were used to analyze the methylation pattern of four Xenopus laevis genes in DNA of embryos, of erythrocytes, and of untreated and estrogen-treated hepatocytes. Within these four genes all sites tested are fully modified in embryonic DNA. However, the adult beta 1-globin gene is unmethylated in DNA of erythrocytes, where it is expressed, and the 68 kd albumin gene, active only in hepatocytes, is specifically hypomethylated in hepatic DNA. The vitellogenin genes A1 and A2, in hepatocytes simultaneously expressed upon estrogen treatment, are heavily methylated in all adult tissues, irrespective of expression. Our results reveal that specific genes can be actively transcribed even when they are fully methylated and that changes in the methylation pattern are not a general prerequisite for gene activation.

Animals↗

PubMed1982

Abnormal RNA splicing causes one form of alpha thalassemia.

The alpha 2-globin gene from a patient with alpha thalassemia contains a pentanucleotide deletion in intron 1 immediately adjacent to exon 1. We tested the functional consequences of this mutation by introducing the thalassemic gene, along with its normal counterpart as a control, into cultured monkey cells on SV40 plasmid vectors. Both genes are expressed, at similar levels, into globin RNA with the correct 5' and 3' ends. However, while most of the normal transcripts are appropriately processed, the thalassemic transcripts are abnormally spliced from a 5' donor site in the middle of exon 1 to the normal 3' acceptor site. This results in the synthesis of a truncated RNA incapable of encoding a normal globin polypeptide. The alternative donor, also used at a low level in monkey cells transfected with the normal gene, shows strong homology to the consensus donor sequence characteristic of many eucaryotic splice junctions. No unspliced or partially spliced thalassemic RNA was detected, indicating that recognition of this site is efficient and does not block removal of intron 2. The alternatively spliced RNA was also found in bone marrow RNA from the alpha-thalassemic patient, although not in that from a normal individual or a beta +-thalassemic patient. The thalassemic phenotype of the patient therefore results from abnormal RNA splicing owing to the deletion of the first splice donor signal.

Base Sequence↗

PubMed1981

Quantitation of DNase I sensitivity in Xenopus chromatin containing active and inactive globin, albumin and vitellogenin genes.

The disappearance of defined restriction fragments of the beta 1-globin, an albumin and the A1 vitellogenin gene was quantitated after DNase I digestion and expressed by a sensitivity factor defined by a mathematical model. Analysis of naked DNA showed that the gene fragments have similar but not identical sensitivity factors. DNase I digestion of chromatin revealed for the same gene fragments sensitivity factors differing over a much wilder range. This is correlated to the activity of the genes analyzed: the beta 1-globin gene fragment is more sensitive to DNase I in chromatin of erythrocytes compared to hepatocytes whereas the albumin gene fragment is more sensitive to DNase I in chromatin of hepatocytes. The A1 vitellogenin gene has the same DNase I sensitivity in both cell types. Comparing the DNase I sensitivity of the three genes in their inactive state we suggest that different chromatin conformations may exist for inactive genes.

Albumins↗

PubMed1981

Estrogen induces tissue specific changes in the chromatin conformation of the vitellogenin genes in Xenopus.

Nuclei from male Xenopus liver were digested extensively with DNase I and the residual amount of the four vitellogenin genes measured by hybridization with a moderate excess of vitellogenin cDNA. The saturation value was about twofold lower in chromatin isolated from liver cells of estrogen treated than from untreated males or from erythrocytes. Analyzing the disappearance of several defined restriction fragments specific for the A1 and A2 vitellogenin genes, after limited digestion with DNase I, suggested that the entire A1 and A2 vitellogenin genes are about twofold more sensitive to DNase I in chromatin of hepatocytes isolated from estrogen treated than from untreated males. Using the same assay no change in the DNase I sensitivity of the two vitellogenin genes in erythrocyte chromatin was observed. Analysis of the beta 1-globin and an albumin gene demonstrated that the DNase I sensitivity of these genes in both cell types is not altered by estrogen. All these data indicate that estrogen stimulation results in an increased DNase I sensitivity specific for the vitellogenin genes in hepatocytes.

Animals↗

PubMed1980

Isolation and translation in vitro of four related vitellogenin mRNAs of estrogen-stimulated Xenopus laevis.

Cloning of vitellogenin cDNA of Xenopus laevis revealed that vitellogenin is encoded in a small family of genes representing two distantly related main groups A and B, each comprising two more closely related subgroups A1, A2, and B1, B2 respectively. To characterize the proteins derived from these genes we have isolated the corresponding mRNAs by hybridizing, under stringent conditions, cytoplasmic poly(a)-containing RNA from the liver of estrogen-stimulated Xenopus to filter-bound cDNA clones containing sequences specific for all four vitellogenin genes. Hybridization of the isolated mRNAs with nick-translated cDNA clones revealed that contamination of the mRNAs by those of the other main group was less than 0.1%. Melting curves of the hybrids prepared with the isolated mRNAs and cDNA clones specific for the four vitellogenin genes showed that the isolated vitellogenin mRNAs are also specific for the four subgroups. Analysis of R loops formed between isolated mRNAs and cDNA clones representing the corresponding subgroup further indicated about 10% cross-contamination between the more closely related mRNAs. In a reticulocyte lysate each of the four mRNAs coded for a 200 000-Mr protein immunoprecipitable by monospecific vitellogenin antibody. From these results we conclude that the four different mRNAs A1, A2, B1 and B2, which all can be isolated efficiently, code for vitellogenin and are expressed simultaneously in response to estrogen stimulation.

Animals↗

PubMed1980

Four different vitellogenin proteins of Xenopus identified by translation in vitro.

Kinetic analysis of vitellogenin mRNA translation in a cell-free reticulocyte lysate translation system revealed that a serine-rich sequence, most probably containing the phosvitin molecule, is located toward the end of the translational product and therefore resides near to the carboxy terminus of the vitellogenin molecule. Translation of the four different vitellogenin mRNAs in vitro and cleavage of the translational products with cyanogen bromide revealed that vitellogenin consists of four different polypeptides, each containing a serine-rich sequence toward its carboxy terminus.

Animals↗

PubMed1978

Estradiol-induced accumulation of vitellogenin mRNA and secretion of vitellogenin in liver cultures of Xenopus.

Explants of male Xenopus liver maintained in a serum-free culture medium respond to stimulation by 2 X 10(-8) M 17beta-estradiol with an increasing rate of accumulation of vitellogenin mRNA, as revealed by hybridization of cDNA to the total cytoplasmic RNA extracted from the cultures. A similar response is observed for secretion of 32PO4-labeled vitellogenin into the culture medium. The in vitro response is improved in liver tissue of prestimulated animals, and by adaptation of liver explants to the culture medium prior to hormone treatment, but attains only about 10% of the in vivo response. Since essential features of the in vivo response are maintained in liver explants, organ culture appears suitable for investigating initial events of estradiol action leading to enhanced synthesis of vitellogenin.

Animals↗

PubMedDate not supplied

Competition for cellular factors that activate metallothionein gene transcription.

Metallothioneins (MTs), small cysteine-rich proteins, bind to and are inducible by heavy metals such as zinc, cadmium and copper. Recent gene-transfer and mutagenesis experiments have elucidated cis-acting DNA sequences involved in this form of regulation, but nothing is known about the trans-acting factors that interact with the control sequences or how such interactions influence the rate of transcription. We report here the detection of cellular factors involved in the cadmium induction of the mouse MT-1 gene by an in vivo competition assay. We show that at least one-class of these cellular factors acts by a positive regulatory mechanism depending on the same region of the 5' flanking DNA required for maximal transcription.

Animals↗