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J Hauber

Publications and source records attributed to J Hauber.

At least 73 records · Page 4Linked to original sources
PubMed1988

Analysis of yeast chromosomal regions carrying members of the glutamate tRNA gene family: various transposable elements are associated with them.

We carried out an analysis on the genomic organisation of the tRNA(Glu) family in S. cerevisiae; eight clones were characterized by restriction mapping, hybridization and sequencing. These data taken together with our earlier findings show that the individual tRNA(Glu3) copies are identical only in their structural part but embedded in entirely different genomic environments. All of the tRNA genes identified here are flanked by elements such as Ty, delta, sigma, and tau. In some cases, sequences from different elements form complex patterns indicating a sophisticated history of these chromosomal regions. A novel observation is that Ty and delta in the regions analyzed are exclusively associated with the tRNA genes. The observed patterns imply that the tRNA genes mark regions of multiple transposition and subsequent excision events, but that these have occurred after the individual tRNA gene copies had been fixed in their present locations. Transcription experiments by the use of micro-injection into Xenopus oocytes suggest that the elements flanking the tRNA genes exert a modulating effect on their expression.

Animals↗
PubMed1988

Functional replacement of the HIV-1 rev protein by the HTLV-1 rex protein.

Two evolutionarily distinct families of human retroviruses, the human immunodeficiency viruses (HIV) and the human T-cell leukaemia viruses (HTLV), have been defined (reviewed in ref. 1). Although these virus groups share tropism for human CD4+ T cells, they differ markedly in primary sequence, genetic organization and disease association (AIDS versus adult T-cell leukaemia), but show similar general strategies for the regulation of viral gene expression. Each encodes a protein able to trans-activate transcription from the homologous viral long terminal repeat (tat in HIV, tax in HTLV), although these proteins act by different mechanisms and do not appear to be interchangeable. Each virus also produces a second trans-acting protein that induces the expression of the unspliced messenger RNAs encoding the viral structural proteins (rev in HIV and rex in HTLV). Here we show that the rex protein of HTLV-I can functionally replace the rev protein of HIV-1 in transient expression assays. This genetic complementation by rex is adequate for the rescue of a replication-defective rev mutant of HIV-1. This unexpected shared function between the structurally distinct rex and rev proteins emphasizes the importance of this highly conserved pathway for the regulation of human retrovirus gene expression.

Gene Expression Regulation↗
PubMed1988

Immunodeficiency virus rev trans-activator modulates the expression of the viral regulatory genes.

The pathogenic human retrovirus human immunodeficiency virus type 1 (HIV-1) encodes two trans-acting nuclear proteins, tat and rev, whose functional expression is essential for viral replication in vitro. The tat protein greatly enhances the expression of both structural and regulatory genes of HIV-1 (linked to the viral long-terminal-repeat promoter element), whereas the rev gene product (previously termed art or trs) has only been shown to be required for the synthesis of structural proteins. Here, we demonstrate that rev also moderates the expression of regulatory genes of HIV-1. It decreases the expression of messenger RNAs that encode the full-length form of the viral tat gene product or the rev protein itself, and induces the synthesis of a previously unreported, truncated tat protein. These actions of rev are mediated by a dramatic shift in the ratio of spliced to unspliced cytoplasmic HIV-1 mRNA. Therefore rev not only activates the synthesis of the viral structural proteins, but also modulates the level and quality of HIV-1 regulatory gene expression.

Gene Expression Regulation↗
PubMed1988

COOH-terminal requirements for the correct processing of a phosphatidylinositol-glycan anchored membrane protein.

Placental alkaline phosphatase (PLAP) is anchored to the plasma membrane by a phosphatidylinositol-glycan (PI-G) moiety. During processing of nascent PLAP, a 29-residue COOH-terminal peptide is cleaved out and the PI-G moiety is attached to the newly created COOH terminus of the mature protein. To investigate the structural requirements of the COOH terminus of the nascent protein for PI-G tailing and anchoring to the plasma membrane, we have transfected COS cells with wild type and mutant forms of cDNA encoding human prepro-PLAP. Utilizing a series of COOH-terminal deletion mutants of prepro-PLAP, it was found that to be PI-G-tailed the newly synthesized protein must possess an uncharged, predominantly hydrophobic amino acid sequence of a minimal length in the COOH-terminal peptide. While forms of prepro-PLAP with 17 consecutive hydrophobic residues in the terminal sequence yielded PI-G-tailed and membrane-bound products, prepro-PLAP mutants with 13 or fewer of such residues yielded hydrophilic proteins that were no longer PI-G-tailed but efficiently secreted into the medium. Studies using cassette mutants demonstrated that the precise amino sequence of the COOH-terminal region could be altered as long as minimal hydrophobicity and length was maintained.

Alkaline Phosphatase↗
PubMed1988

Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells.

This paper describes a novel eukaryotic reporter gene, secreted alkaline phosphatase (SEAP). In transient expression experiments using transfected mammalian cells, we demonstrate that SEAP yields results that are qualitatively and quantitatively similar, at both the mRNA and protein levels, to parallel results obtained using established reporter genes. However, SEAP offers significant advantages in terms of ease of assay and assay expense, and also has the potential for quantitative assay at levels as low as 0.2 pg/ml of culture medium. These attributes suggest that SEAP may have general utility in experiments which rely on the accurate measurement of reporter gene expression levels.

Alkaline Phosphatase↗
PubMed1988

Phosphorylation of the rev gene product of human immunodeficiency virus type 1.

Replication of human immunodeficiency virus type 1 requires the functional expression in trans of the virally encoded rev gene product (previously called art/trs). Here we demonstrate that this protein can be metabolically labeled with 32Pi. The phosphate receptor in the rev protein is shown to be exclusively serine. Treatment of rev-expressing cells with phorbol ester, a specific activator of protein kinase C, led to significant but transient enhancement of the level of rev phosphorylation. These results indicate that the rev protein is posttranslationally modified in vivo and suggest that the level of this modification is subject to modulation by extracellular stimuli.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

Mutational analysis of the trans-activation-responsive region of the human immunodeficiency virus type I long terminal repeat.

We used site-directed mutagenesis to delineate sequences within the human immunodeficiency virus type I (HIV-I) long terminal repeat (LTR) required for trans-activation by the viral tat gene product. We demonstrated that sequences 3' to LTR position +44 are dispensable for trans-activation but that almost all of the mutations tested located between positions -17 and +44 greatly reduced trans-activation at both the transcriptional and posttranscriptional levels. However, displacement of the HIV-I LTR trans-activation-responsive region (TAR) 3' by insertion of up to 32 base pairs between the LTR TATA box and cap site had little effect on trans-activation. An analysis of the DNase I hypersensitivity profile of the HIV-I LTR in transfected cultures suggested the presence of at least two DNase I-hypersensitive sites, including one which extends into the viral TAR element; however, neither of these sites appeared to be significantly affected by tat coexpression. These results allow more precise delineation of the sequences important for TAR function and suggest that the TAR may be recognized by a host-specific DNA-binding protein rather than by the tat protein directly.

Base Sequence↗
PubMed1988

Subcellular localization of the human immunodeficiency virus trans-acting art gene product.

The genome of the human immunodeficiency virus is distinguished from other animal retroviruses by the presence of several additional open reading frames. The protein product of one of these novel genes, which has been termed art or trs, is required for the expression of the virus structural genes but not for the expression of virus encoded regulatory proteins. Immunocytochemistry and subcellular fractionation demonstrate that the art protein is located predominantly in the nucleus. Therefore, any proposed mechanism for the function of art is likely to involve nuclear events.

Animals↗
PubMed1987

One member of the tRNA(Glu) gene family in yeast codes for a minor GAGtRNA(Glu) species and is associated with several short transposable elements.

During characterization of the whole tRNA-(Glu) family from the yeast, Saccharomyces cerevisiae, we isolated one cosmid clone bearing a tRNA(Glu) gene copy that is deviant from the major tRNA(Glu3) gene members in only five positions. This divergent tRNA-(Glu) is a minor species and is represented by a single gene copy. One of the nucleotide exchanges concerns the anticodon which is modified from T-T-C in the tRNA(Glu3) gene to C-T-C which implies that this tRNA serves the codon triplet G-A-G. Two other minor yeast tRNA species have been reported which appear to be particularly designed for the translation of those codons that have a G in its third (Wobble) position. The low abundance of such minor tRNA species correlates positively to the low occurrence of most of the N-N-G codons in yeast. Furthermore, the GAGtRNA-(Glu) locus represents another case of the general phenomenon in which the majority of the tRNA genes in yeast are associated with one or several transposable elements forming complex patterns. In this particular case, divergent segments of delta and tau are present in the 5' flanking region of the tRNA gene and arranged in a novel configuration. The sequence data lend support to the view that tau is not an evolutionary young element as was earlier anticipated.

Base Sequence↗
PubMed1987

Trans-activation of human immunodeficiency virus gene expression is mediated by nuclear events.

Human immunodeficiency virus encodes a gene product termed tat that is able to activate viral gene expression when present in trans. The mechanism of action of the tat gene product appears to be bimodal, resulting in both an increase in the steady-state level of viral mRNA and the enhanced translation of that RNA. In this report we have examined the mechanism by which tat elevates viral mRNA levels. Data are presented demonstrating that tat acts by increasing the rate of viral transcription, rather than by modulating the stability of viral mRNA. Indirect immunofluorescence was used to show that tat is predominantly localized in the nucleus of expressing cells, a location consistent with a role in the regulation of viral transcription. These results suggest that tat could play a role in human immunodeficiency virus replication essentially similar to that proposed for the trans-acting nuclear gene products described for several other virus species.

Cell Compartmentation↗
PubMed1986

Conserved and non-conserved features among the yeast Ty elements.

We have isolated and characterized a Ty element from a yeast cosmid library which exhibits several unusual features: it is flanked by non-homologous delta elements and directly associated with a singular delta element. A tRNA(Glu3) gene and tRNA(Cys) gene are found in conjunction with this element, located in opposite orientation on either end of it. The sequence information now available for several Ty elements has been used in a detailed comparative analysis to determine conserved features among the Ty elements, preferably between class I elements and a class II element. Highly conserved sequence motifs appear to be located at the borders of particular segments that correspond to the putative protein domains of the Tys. Furthermore, we include a comparison of the best-conserved amino acid homologies for these putative proteins of Ty elements, transposable elements from other organisms and several retroviral proviruses to confirm their close structural resemblance.

Amino Acid Sequence↗
PubMed1986

Enhancer-like stimulation of yeast tRNA gene expression by a defined region of the Ty element micro-injected into Xenopus oocytes.

In the yeast Saccharomyces cerevisiae, the majority of the tRNA genes are found associated with transposable elements one of which is the Ty element. We noticed that the transcriptional activity of several of these genes was rather different depending on the type of the accompanying element(s) [Nelböck et al. (1985) Biol. Chem. Hoppe-Seyler 366, 1041-1051]. In order to study the influence of a Ty element on tRNA gene expression, we used the method of micro-injection into Xenopus oocytes taking advantage of a Ty+/Ty- allelic pair of a tRNALys1 gene recently isolated in our laboratory. A direct comparison showed that the expression of the plus allele was c. sixfold higher than that of the minus allele. In order to determine sequences of the Ty responsible for this effect, we constructed a number of variants in which distinct segments of the Ty were deleted or rearranged. The activating ability was localized to a 590-bp segment of the Ty containing the 'promoter delta', independent of its orientation or location towards the tRNA gene thus resembling the effects described for transcriptional enhancers. This is the first time that a long-range effect has been observed on the expression of a gene transcribed by RNA polymerase III.

Animals↗
PubMed1985

Nucleotide sequence and characteristics of a Ty element from yeast.

We have determined the nucleotide sequence of a complete yeast Ty element (Ty-pY109) which is located near a tRNA(Lys1) gene. The element is 5912 bp in length; the internal domain is flanked by two identical delta sequences of 331 bp. Ty-pY109 contains two large open reading frames (ORFs) which overlap by 38 bp; the putative proteins consist of 440 and 1328 amino acid residues, respectively. The organisation of the coding sequences in Ty resembles that found in retroviral proviruses and the copia-like elements in Drosophila. Partial homologies have been found between Ty-ORF1 and tnpA from Tn3, and Ty-ORF2 and a reverse transcriptase-like domain (1,2).

Alleles↗