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Biomedical subjects

F D Bushman

Publications and source records attributed to F D Bushman.

50 records · Page 3Linked to original sources
PubMed1993

The bacteriophage 434 right operator. Roles of O(R)1, O(R)2 and O(R)3.

Lysogenic induction of bacteriophage lambda is controlled by the action of the phage repressor and Cro proteins at the phage right operator (O(R)). This study examines the roles of the repressor and Cro proteins of the related phage 434. The start sites of transcription of the divergently oriented promoters in the 434 O(R) region, PR and PRM, were mapped, and the effects of 434 repressor and Cro on promoter activity were assessed using promoter fusions to lacZ. The effects of repressor or Cro bound to each of the operator subsites (O(R)1, O(R)2 and O(R)3) were assessed by examining regulation in the presence of operator mutations. The binding of Cro to a 434 operator was probed by an ethylation interference experiment which, together with other data, indicates that 434 Cro and repressor probably turn off transcription by blocking binding of RNA polymerase to promoter sequences. In general, the 434 and lambda right operators are controlled in a similar fashion, but differences in detail were also encountered: (1) 434 Cro represses transcription from PR primarily by binding to O(R)1, whereas binding of lambda Cro to O(R)1 and O(R)2 contribute equally to repression. (2) The 434 cI message, unlike that of lambda, has a recognizable homology to the Shine-Dalgarno ribosome binding site. (3) Occupancy of O(R)3 by repressor may be somewhat greater in a 434 lysogen than in a lambda lysogen. (4) The 434 repressor probably activates transcription when bound at O(R)2 by contacting RNA polymerase, as does lambda repressor, but also by influencing competition between PR and PRM. An analysis of the six right operator systems for which data are available indicates that all six repressors may employ the mechanism of transcriptional activation first described for lambda, P22 and 434: apposition of an acidic surface to a particular part of RNA polymerase.

Base Sequence↗
PubMed1993

Identification of discrete functional domains of HIV-1 integrase and their organization within an active multimeric complex.

HIV-1 integrase protein possesses the 3' processing and DNA strand transfer activities that are required to integrate HIV DNA into a host chromosome. The N-, C-terminal and core domains of integrase are necessary for both activities in vitro. We find that certain pairs of mutant integrase proteins, which are inactive when each protein is assayed alone, can support near wild type levels of activity when both proteins are present together in the reaction mixture. This complementation implies that HIV-1 integrase functions as a multimer and has enabled us to probe the organization of the functional domains within active mixed multimers. We have identified a minimal set of functional integrase domains that are sufficient for 3' processing and DNA strand transfer and find that some domains are contributed in trans by separate monomers within the functional complex.

Base Sequence↗
PubMed1992

Integration of human immunodeficiency virus DNA: adduct interference analysis of required DNA sites.

The integration (IN) protein encoded by human immunodeficiency virus directs the integration of viral DNA into host DNA. We have probed the DNA sites required for the function of IN protein by attaching adducts to model DNA substrates and assaying their effects on integration in vitro. These experiments reveal that modifications in a short region on both DNA strands at the ends of the viral DNA block IN protein function. Modification of the target DNA near the point of DNA strand transfer also blocks IN protein function. Further experiments suggest that distinct subsets of the identified interactions are important for separate steps in the integration process.

Autoradiography↗
PubMed1991

A rapid in vitro assay for HIV DNA integration.

Retroviruses synthesize a double stranded DNA copy of their RNA genome after infection of a permissive cell and subsequent integration of this DNA copy into the host genome is necessary for normal viral replication. Integration occurs by a specialized DNA recombination reaction, mediated by the viral IN protein. Because this reaction has no known cellular counterpart, it is a particularly attractive target in the search for specific inhibitors with low toxicity that may serve as therapeutic antiviral agents. We present a simple assay system that is suitable for screening potential inhibitors of HIV DNA integration. Only short oligonucleotides matching one end of HIV DNA and purified HIV IN protein are required as substrates. Furthermore, since each step of the assay can be carried out in the wells of microtiter plates, large numbers of reactions can be processed simultaneously.

Animals↗
PubMed1991

Activities of human immunodeficiency virus (HIV) integration protein in vitro: specific cleavage and integration of HIV DNA.

Growth of human immunodeficiency virus (HIV) after infection requires the integration of a DNA copy of the viral RNA genome into a chromosome of the host. Here we present a simple in vitro system that carries out the integration reaction and the use of this system to probe the mechanism of integration. The only HIV protein necessary is the integration (IN) protein, which has been overexpressed in insect cells and then partially purified. DNA substrates are supplied as oligonucleotides that match the termini of the linear DNA product of reverse transcription. In the presence of HIV IN protein, oligonucleotide substrates are cleaved to generate the recessed 3' ends that are the precursor for integration, and the cleaved molecules are efficiently inserted into a DNA target. Analysis of reaction products reveals that HIV IN protein joins 3' ends of the viral DNA to 5' ends of cuts made by IN protein in the DNA target. We have also used this assay to characterize the sequences at the ends of the viral DNA involved in integration. The assay provides a simple screen for testing candidate inhibitors of HIV IN protein; some such inhibitors might have useful antiviral activity.

Base Sequence↗
PubMed1990

Retroviral DNA integration directed by HIV integration protein in vitro.

Efficient retroviral growth requires integration of a DNA copy of the viral RNA genome into a chromosome of the host. As a first step in analyzing the mechanism of integration of human immunodeficiency virus (HIV) DNA, a cell-free system was established that models the integration reaction. The in vitro system depends on the HIV integration (IN) protein, which was partially purified from insect cells engineered to express IN protein in large quantities. Integration was detected in a biological assay that scores the insertion of a linear DNA containing HIV terminal sequences into a lambda DNA target. Some integration products generated in this assay contained five-base pair duplications of the target DNA at the recombination junctions, a characteristic of HIV integration in vivo; the remaining products contained aberrant junctional sequences that may have been produced in a variation of the normal reaction. These results indicate that HIV IN protein is the only viral protein required to insert model HIV DNA sequences into a target DNA in vitro.

Animals↗
PubMed1990

Sequence requirements for integration of Moloney murine leukemia virus DNA in vitro.

Normal replication of Moloney murine leukemia virus (MoMLV) requires the integration of a DNA copy of the viral RNA genome into a chromosome of the host. In this work, we characterize the DNA sequences at the ends of the linear proviral precursor that are required for integration in the presence of MoMLV integration protein in vitro. We found that nine bases of MoMLV DNA at each end of a linear model substrate were sufficient for near-maximal levels of integration and that four bases of MoMLV DNA at each end were sufficient for low levels of correct integration. We also found that a 3'-terminal A residue was preferred for integration. We infer from the limited DNA sequence requirements for integration that factors in addition to DNA sequence direct integration protein to act at the ends of the viral DNA.

Base Sequence↗
PubMed1989

A single glutamic acid residue plays a key role in the transcriptional activation function of lambda repressor.

Previous experiments have suggested that negative charge is an important aspect of the activating region of lambda repressor as it is for at least one class of eukaryotic transcriptional activators. Here we randomize amino acids in the activating region of repressor and assay the function of over 100 variants. We find that acidic residues at the four solvent-exposed positions on the surface of an alpha helix (helix 2 in the structure) together comprise a strong activating region. Only one of these acidic residues, however, is critical for activation, and at this position glutamate is strongly preferred to aspartate. At the three remaining positions, certain uncharged residues (different ones at each position) function as well as or better than the acidic residues. Basic residues, however, are highly detrimental to function at all four positions. Our mutagenesis studies also suggest limitations on amino acid substitutions that allow formation of the helix-turn-helix DNA binding motif found in repressor and in many other DNA binding regulatory proteins.

Amino Acid Sequence↗
PubMed1988

Turning lambda Cro into a transcriptional activator.

According to our present understanding, lambda repressor bound to DNA stimulates transcription by touching RNA polymerase bound at an adjacent promoter. The part of repressor required for activation was identified in part by the isolation of mutants specifically impaired in transcriptional activation. The amino acids of repressor altered in these "positive control" mutants lie in an acidic patch on the surface of repressor that is closely apposed to RNA polymerase. In this study, we show that this "activating patch" of repressor is sufficient for transcriptional activation in another sequence context. We transfer this activating patch onto the surface of lambda Cro, a protein normally unable to activate transcription, and show that the modified Cro is a transcriptional activator. In addition, we provide evidence that the repressor protein of phage 434 also activates transcription using an activating patch similar to that of lambda repressor.

Bacteriophage lambda↗
PubMed1986

Activation of transcription by the bacteriophage 434 repressor.

Bacteriophage 434 encodes a repressor that, like bacteriophage lambda repressor, both activates and represses transcription. As in the lambda chromosome, a region of the 434 chromosome, called the right operator, contains three repressor binding sites (OR1, OR2, and OR3) that mediate these effects on two adjacent promoters. We now show that a part of the 434 repressor, the amino-terminal domain, activates leftward transcription when bound to OR2. We show that 434 repressor bound to OR2 closely approaches (touches) RNA polymerase bound to the leftward promoter. Model building based on ethylation interference and other experiments suggests that in three cases, those involving lambda repressor, 434 repressor, and bacteriophage P22 repressor, and in spite of differences in detailed arrangements, transcription is activated by a contact between the repressor and the same part of RNA polymerase.

Coliphages↗
PubMed1983

Transcripts of paternal and maternal actin gene alleles are present in interspecific sea urchin embryo hybrids.

Analysis of actin-coding RNAs in interspecific hybrid sea urchin embryos of Strongylocentrotus purpuratus and Lytechinus variegatus, and S. purpuratus and S. droebachienis has revealed the presence of transcripts from both paternal and maternal S. purpuratus actin gene alleles. In the L. variegatus female X S. purpuratus male embryos transcripts from at least two different paternal actin gene alleles are present in both the blastula and prism stages. In the reciprocal S. purpuratus female X L. variegatus male embryos, the same two maternal (S. purpuratus) alleles were also expressed as RNA in blastula. The S. droebachiensis female X S. purpuratus male embryos appear to contain transcripts from at least one paternal actin gene allele at the blastula stage. The paternally derived actin-coding RNAs are the same size as the mature actin mRNAs expressed in normal S. purpuratus embryos. Since all known S. purpuratus actin genes contain at least two introns, the paternal alleles are not only transcribed in the hybrid embryos, but also the primary transcripts are probably processed to mature mRNA. An explanation of the diversity of observations in the literature on paternal genome expression in hybrid sea urchin embryos is discussed.

Actins↗
PubMed1983

Conserved pattern of embryonic actin gene expression in several sea urchins and a sand dollar.

An examination of the size and relative abundance of actin-coding RNA in embryos of four sea urchins (Strongylocentrotus purpuratus, Strongylocentrotus droebachiensis, Arbacia punctulata, Lytechinus variegatus) and one sand dollar (Echinarachnius parma) reveals a generally conserved program of expression. In each species the relative abundance of these sequences is low in early embryos and begins to rise during late cleavage or blastula stages. In the four sea urchins, actin-coding RNAs increase between approximately 9- and 35-fold by pluteus or an earlier stage, and in the sand dollar about 5.5-fold by blastula. A major actin-coding RNA class of 2.0-2.2 kilobases (kb) is found in each species. A smaller actin-coding RNA class, which accumulates during embryogenesis, is also present in S. purpuratus (1.8 kb), S. droebachiensis (1.9 kb), and A. punctulata (1.6 kb), but apparently absent in L. variegatus and E. parma. In S. droebachiensis, actin-coding RNA is relatively abundant in unfertilized eggs and drops sharply by the 16-cell stage. This is in contrast to the other sea urchins where the actin message content is relatively low in eggs and does not change substantially in the embryos throughout early cleavage. The observations in this study suggest that the pattern of embryonic expression of at least some members of this gene family is ancient and conserved.

Actins↗
PubMedDate not supplied

Ethylation interference and X-ray crystallography identify similar interactions between 434 repressor and operator.

In the crystal structure of a repressor-operator complex (the 434 repressor DNA-binding domain and its 14-base pair (bp) operator), Anderson et al. elsewhere in this issue identify six positions of likely contact between repressor protein and phosphates of the DNA backbone. At each of these positions, electron densities of protein and DNA merge. Experiments presented here indicate that intact 434 repressor approaches these phosphates very closely when it is bound to DNA in solution. Specifically, when any one of these phosphates is ethylated, repressor cannot bind to the modified operator. We also identify another position where ethylation has a significant but less dramatic effect on repressor binding, and note that in the structure, repressor closely approaches this phosphate. Our results strongly support the idea that the interactions between protein and the DNA phosphate backbone in the crystallized complex are the same as those made by intact repressor to operator DNA in solution. In addition, our results suggest that DNA is slightly bent by repressor binding.

Alkylation↗