Subregions of a conserved part of the HIV gp41 transmembrane protein are differentially recognized by antibodies of infected individuals.

Certa, Ulrich; Bannwarth, W.; Stüber, Dietrich; Gentz, Reiner; Lanzer, M; Grice, Stuart Le; Guillot, F; Wendler, I.; Hunsmann, Gerhard; Bujard, Hermann

doi:10.1002/j.1460-2075.1986.tb04605.x

Cited by 126 publications

(64 citation statements)

References 35 publications

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“…Following all mutagenesis and reconstruction, each nuclcotidc sequence wz confn-mcd by didcoxy sequencing. E, cofi strain M 15 w;is used throughout for expression st udics [7].…”

Section: Methodsmentioning

confidence: 99%

Intrinsic activity of precursor forms of HIV‐1 proteinase

et al. 1992

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The wild-type -Phe*Pro-bond located at the N-terminus of the mature aspnrtic protcinasc of HIV-l was repluccd by -llc-Pro-or -V&Pro-. By this means, processing at this clcavngc junction was prevented and so. extended or precursor fcxs of HIV-protcinase were gcncmted. Thcsc constructs were expressed in ~xchcricl~iu cofi. purified therefrom, and their specificity. activity ut different pH values and susceptibility to the potent inhibitor, Ro3 l-8959, was assessed, A hitherto unobserved cleavage junction (at -Alu-Phe*Lcu-Gin-)in the frame-shift region of the gag-p01 viral genome was identified and contirmcd by demonstrating clcnvagc of a synthetic pcptide corresponding to this region. The implications for viral replication of self-processing at neutral pH by proteinase whilst still present (in a prccuisur tjrm) as a component of the polyprotcin arc considered; such reactions, however, are still blocked cvcn at pH values us high us 8.0 by Ro3 l-8959.HIV-proteinase precursor; Activity and pH dcpcndcnce; Inhibition by Ro31-8959; Novel polyprotein clcuvagc junction

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“…Following all mutagenesis and reconstruction, each nuclcotidc sequence wz confn-mcd by didcoxy sequencing. E, cofi strain M 15 w;is used throughout for expression st udics [7].…”

Section: Methodsmentioning

confidence: 99%

Intrinsic activity of precursor forms of HIV‐1 proteinase

et al. 1992

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“…The inactivation of the right IR of the element by the introduction of a new EcoRI site at 1216 bp (Olasz et al, manuscript submitted) and subsequent cloning steps resulted in pJKI110, in which the right IR was partially deleted (small black triangle). The insertion of the lacl ~ gene (dotted rectangle) from pDMI,1 [13] into pJKI110 yielded pJKI132. In pJKI154 the tac promoter was removed and the left IR was truncated (small white triangle), pJKI154 contained the IS30 sequence from the unique Hincll site to the newly introduced EcoRI site (bp 17 1216).…”

Section: The Measurement Of the In Trans Activity Of Ls30mentioning

confidence: 99%

The construction and characterization of an effective transpositional system based on IS30

1996

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We constructed an in vivo system to detect transpositional rearrangements induced by the insertion sequence IS30. The transposase protein expressed from the transposase producer plasmids catalyzed rearrangements on different target sequences presented in trans. High yields, up to 83%, of transpositional frequencies were observed. The frequency of rearrangements correlated with the amount of transposase protein produced and the attractivity of the target sequences. Alteration in the frequency of transposition was observed in the recA-E. coli strains JM109 and TG2. Remarkable structural and functional analogy was found with site-specific recombination systems.Key words: IS30; Overproduction of IS30 transposase; In vivo assay; Transpositional frequency; Escherichia coli by the genomic copies of the element) is too low to carry out transpositional reactions effectively; (2) the transposase protein can act only in cis, i.e. on the same replicon from which it was produced -as was shown in the case of Tn5 [7][8][9].To clarify this question we studied the in trans activity of the IS30 transposase in reactions characteristic of the element, and investigated the correlation between the expression level of IS30 transposase and the transpositional frequency. In our experiments the IS30 transposase was able to perform sitespecific and 'classical' transpositional reactions in trans (dimerization and deletion, respectively), at very high frequencies, up to 82.7%. The system described here represents a simple and useful tool in the isolation and characterization of mutants and in the development of an in vitro system.

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“…Plasmid pDlOGal4(1-94) and bacterial strain MC1061/PDMI,1 for expression of H6Gal4(1-94) were generously provided by C. Rosen. This expression system is derived from the pDS expression plasmid (7,10,35 Fig/ml, respectively) were inoculated with 2 ml of an overnight culture. Induction was started at an optical density at 580 nm of 0.4 by addition of 1 mM (final concentration) IPTG.…”

Section: Constructionmentioning

confidence: 99%

Definition of the transcriptional activation domain of recombinant 43-kilodalton USF.

Kirschbaum¹,

Pognonec²,

Roeder³

1992

Mol. Cell. Biol.

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The cellular transcription factor USF is involved in the regulation of both cellular and viral genes and consists of 43-and 44-kDa polypeptides which independently show site-specific DNA binding. Cloning of the corresponding cDNA revealed that the 43-kDa polypeptide (USF43) is a member of the basic (B)-helix-loop-helix (HLH)-leucine zipper (LZ) family of proteins and provided a means for its functional dissection. Initial structure-function studies revealed that the HLH and LZ regions are both important for USF43 oligomerization and DNA binding. The studies presented here have focused on the determination of domains that contribute to transcriptional activation in vitro and show that (i) both a small region close to the N terminus and a region between residues 93 and 156 contribute strongly to transcriptional activation, (ii) full activation depends on the presence of both domains, (iii) the B-HLH-LZ region has no intrinsic activation potential but DNA binding is absolutely required for transcriptional activation, and (iv) the B-HLH-LZ region can be replaced by the Gal4 DNA binding domain without loss of activation potential.The ubiquitous cellular transcription factor USF is a member of the helix-loop-helix (HLH) family of regulatory proteins. USF was originally described as an upstream stimulatory factor that binds to the core sequence CACGTG in the adenovirus major late promoter (5,9,22,23,31). It subsequently was shown to be involved in the regulation of a number of cellular genes, including those encoding mouse metallothionein I (6), rat y-fibrinogen (8), human growth hormone (25), and Xenopus TFIIIA (15,33). USF was also shown to interact with the essential ,uE3 motif in the immunoglobulin heavy-chain enhancer (1) as well as with a pyrimidine-rich initiator element found in many genes (30). Together with the further demonstration of cooperative interactions between USF and the initiator-binding protein TFII-I (30), these findings raised the possibility of a more general involvement of USF in transcriptional regulation.Purified human USF is composed of 43-and 44-kDa polypeptides (USF43 and US"4") which show independent site-specific DNA binding (32 (28) and thus can be used for further functional dissection. The objective of this study was to determine the activation domain(s) within human USF43. A major obstacle to an analysis of this question in vivo is the high level of endogenous USF in most or all cell types, making the results of transfection experiments difficult to interpret. To circumvent this problem, we used an in vitro transcription system which consisted of nuclear extracts that were immunodepleted of endogenous USF and showed only basal-level transcription (28). In this system, exogenous rUSF43 elevated transcription from a USF-responsive promoter (a human immunodeficiency virus [HIV] core promoter with two upstream USF sites) to the level observed in unfractionated nuclear extracts. The analysis of mutated forms of USF43 allowed the identification of two regions N terminal of the B-HLH-LZ r...

show abstract

Subregions of a conserved part of the HIV gp41 transmembrane protein are differentially recognized by antibodies of infected individuals.

Cited by 126 publications

References 35 publications

Intrinsic activity of precursor forms of HIV‐1 proteinase

Intrinsic activity of precursor forms of HIV‐1 proteinase

The construction and characterization of an effective transpositional system based on IS30

Definition of the transcriptional activation domain of recombinant 43-kilodalton USF.

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