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Biomedical subjects

J Karn

Publications and source records attributed to J Karn.

At least 19 recordsLinked to original sources
PubMed1995

The structure of the human immunodeficiency virus type-1 TAR RNA reveals principles of RNA recognition by Tat protein.

The human immunodeficiency virus type-1 (HIV-1) Tat protein stimulates transcriptional elongation. Tat is introduced to the transcription machinery by binding to the transactivation response region (TAR) RNA stem-loop encoded by the 5' leader sequence found on all HIV-1 mRNAs. We have used multidimensional heteronuclear NMR to determine the structure of the TAR RNA in the presence of the ADP-1 polypeptide, a 37-mer that carries the minimal RNA recognition region of the Tat protein and closely mimics Tat binding specificity. In the presence of a variety of ligands, including ADP-1, related basic peptides and the amino acid derivative argininamide, the bulge region of TAR undergoes a local conformational rearrangement and forms a more stable structure. The structure of TAR in the bound form has been determined from over 1000 NMR-derived constraints. The U23 residue at the 5' end of the bulge is positioned near G26 and A27 in the major groove, rather than stacked on A22 as in the free TAR. U23 and G26 are brought into close proximity by contacts to the guanidinium group and side-chain amide group of a common arginine residue. However, the interaction of this guanidinium group with TAR is not the only source of binding specificity. Besides NOEs to the arginine residue participating in the conformational change, ADP-1 shows additional intermolecular NOEs to TAR, suggesting that there are multiple points of contacts between TAR RNA and residues from the basic and core regions of Tat. These structural results provide important clues towards the identification of small molecular mass and/or peptidomimetic inhibitors of the essential Tat-TAR interaction.

Amino Acid Sequence↗
PubMed1995

Progress in anti-HIV structure-based drug design.

The course of drug development for the treatment of HIV-1 infection and AIDS is being revolutionized by high-resolution structures of essential viral proteins. We survey the impact on drug design of the recently elucidated structural knowledge of two essential enzymes, reverse transcriptase and protease, and three new targets, the viral integrase and the gene regulatory protein-RNA interactions, Tat-TAR and Rev-RRE.

Antiviral Agents↗
PubMed1993

HIV gene regulatory proteins tat and rev and their interactions with synthetic RNA.

Synthetic oligoribonucleotides have been prepared and annealed to form model RNA duplexes that mimic the high affinity RNA recognition sites for the HIV-1 tat and rev proteins. The contributions of individual functional groups on the model RNAs to the specificity of binding by their respective proteins were studied by use of oligoribonucleotides containing site-specifically modified nucleotides. Both tat and rev appear to recognise specifically a limited number of functional groups in the major groove of an RNA double helix distorted by virtue of unpaired or non-Watson-Crick paired nucleotides.

Base Sequence↗
PubMed1992

Recognition of the high affinity binding site in rev-response element RNA by the human immunodeficiency virus type-1 rev protein.

The Human Immunodeficiency Virus type-1 rev protein binds with high affinity to a bubble structure located within the rev-response element (RRE) RNA in stemloop II. After this initial interaction, additional rev molecules bind to the RRE RNA in an ordered assembly process which requires a functional bubble structure, since mutations in the bubble sequence that reduce rev affinity block multiple complex formation. We have used synthetic chemistry to characterize the interaction between rev protein and its high affinity binding site. A minimal synthetic duplex RNA (RBC6) carrying the bubble and 12 flanking base pairs is able to bind rev with 1 to 1 stoichiometry and with high affinity. When the bubble structure is inserted into synthetic RNA molecules carrying longer stretches of flanking double-stranded RNA, rev forms additional complexes resembling the multimers observed with the RRE RNA. The ability of rev to bind to RBC6 analogues containing functional group modifications on base and sugar moieties of nucleoside residues was also examined. The results provide strong evidence that the bubble structure contains specific configurations of non-Watson--Crick G:G and G:A base pairs and suggest that high affinity recognition of RRE RNA by rev requires hydrogen bonding to functional groups in the major groove of a distorted RNA structure.

Bacteriophage T7↗
PubMed1991

Human immunodeficiency virus type 1 regulator of virion expression, rev, forms nucleoprotein filaments after binding to a purine-rich "bubble" located within the rev-responsive region of viral mRNAs.

The human immunodeficiency virus type 1 rev protein binds with high affinity (Kd less than 1-3 nM) to a purine-rich "bubble" containing bulged GG and GUA residues on either side of a double-helical RNA stem-loop located toward the 5' end of rev-response element RNA. High-affinity rev binding is maintained when the bubble is placed in heterologous stem-loop structures, but rev binding is reduced when either the bulged residues or flanking base pairs in the stem are altered. Rev binding to the purine-rich bubble nucleates assembly of long filamentous ribonucleoprotein structures containing polymers of rev bound to flanking RNA sequences. It is proposed that rev regulates human immunodeficiency virus RNA expression by selectively packaging viral transcripts carrying the rev-response element sequence into rod-like nucleoprotein complexes that block splicing of the packaged mRNAs.

Base Sequence↗
PubMed1991

RNA binding by the tat and rev proteins of HIV-1.

HIV-1 tat protein binds specifically to HIV-1 TAR RNA. A Scatchard analysis of tat binding has shown that the purified protein forms a one-to-one complex with HIV-1 TAR RNA with a dissociation constant of Kd = 12 nM. Tat binding in vitro is dependent upon the presence of 3 non-base paired U residues which produce a 'bulge' in the TAR RNA stem-loop structure. Deletion of the uridine residues in the bulge or substitution with guanine residues produced RNAs with a 6 to 8-fold lower affinity than wild-type TAR. By contrast, mutations that alter the sequence of the 6 nucleotide-long loop at the tip of TAR RNA structure, and mutations which alter the sequence of the stem whilst preserving Watson-Crick base pairing, do not affect tat binding significantly. There is a direct correlation between the ability of tat to bind to TAR RNA and to activate HIV transcription. Viral LTRs encoding TAR sequences known to bind tat weakly, are not stimulated efficiently by tat in vivo. HIV-1 regulator of virion expression (rev) protein binds specifically to RNA transcripts containing the 223 nucleotide-long RRE sequence with an apparent dissociation constant of 1-3 nM. The minimum binding site for rev is a 'bubble' containing 2 G residues on one side and the sequence AGU on the other. Rev is able to bind efficiently to this restricted site in the context of the RRE sequence as well as in the context of a stable RNA duplex with a sequence unrelated to that found in the RRE.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

Control of human immunodeficiency virus replication by the tat, rev, nef and protease genes.

Immediately after infection, human immunodeficiency virus directs the synthesis of three regulatory proteins tat, rev and nef that together allow the synthesis of the structural proteins of the virus after a delay of several hours. Viral mRNA production is controlled by the tat gene, which appears to stimulate elongation by RNA polymerase II, and the rev gene, which allows the accumulation of unspliced or partially spliced mRNAs in the cytoplasm. The nef gene is dispensible for virus growth but may limit virus spread by downregulating the levels of cellular surface proteins such as the CD4 receptor. Virus maturation also depends critically on the protease gene which allows the orderly rearrangement of the viral core structures in newly budded virions as well as the vpu and vif genes which allow efficient production of mature envelope glycoprotein.

Chromosome Mapping↗
PubMed1990

HIV-1 regulator of virion expression (Rev) protein binds to an RNA stem-loop structure located within the Rev response element region.

HIV-1 Rev protein, purified from E. coli, binds specifically to an RNA transcript containing the 223 nucleotide long Rev response element (RRE) sequence. Rev binds to RRE in vitro with an apparent dissociation constant of 1 to 3 nM as determined by filter binding, gel mobility shift assays, or an immunoprecipitation assay using a monoclonal antibody specific for the Rev C-terminus. Antisense RRE sequences are bound by Rev with a 20-fold lower affinity than wild-type RRE sequences. The Rev-RRE complex forms even in the presence of a 10,000-fold molar excess of 16S rRNA, whereas formation of the low affinity antisense RRE-Rev complex is efficiently blocked by addition of excess 16S rRNA. A approximately 33 nucleotide fragment is protected from ribonuclease T1 digestion by the binding of Rev to RRE RNA, suggesting that Rev binds with high affinity to only a restricted region of the RRE. This protected fragment is unable to rebind Rev protein but has been mapped to a 71 nucleotide long Rev binding domain sequence that overlaps the protected fragment.

Amino Acid Sequence↗
PubMed1990

HIV-1 tat protein stimulates transcription by binding to a U-rich bulge in the stem of the TAR RNA structure.

The HIV-1 trans-activator protein, tat, is an RNA binding protein with a high affinity for a U-rich bulge near the tip of the stem in the RNA stem-loop structure encoded by the trans-activation responsive region (TAR). A Scatchard analysis of tat binding has shown that the purified protein forms a one-to-one complex with HIV-1 TAR RNA with a dissociation constant of Kd = 12 nM. Deletion of the uridine residues in the bulge or substitution with guanine residues produced RNAs with a 6- to 8-fold lower affinity than wild-type TAR. Introduction of a point mutation expected to destabilize base pairing in nearby residues of the TAR stem-loop structure reduced tat binding 10-fold. In contrast, mutations that alter the sequence of the six nucleotide long loop at the tip of TAR RNA structure, and mutations which alter the sequence of the stem whilst preserving Watson-Crick base pairing, do not affect tat binding significantly. There is a direct correlation between the ability of tat to bind to TAR RNA and to activate HIV transcription. Viral LTRs carrying TAR sequences encoding any of the mutations known to produce transcripts which bind tat weakly, are not stimulated efficiently by tat in vivo.

Base Sequence↗
PubMed1989

Regulatory and essential light-chain-binding sites in myosin heavy chain subfragment-1 mapped by site-directed mutagenesis.

Site-directed mutagenesis of the cloned subfragment-1 (S-1) region of the unc-54 gene, encoding the myosin heavy chain B (MHC B) from Caenorhabditis elegans, has been used to locate binding sites for the regulatory and essential light chains. MHC B S-1 synthesized in Escherichia coli co-migrated with rabbit skeletal muscle myosin S-1 (Mr 90,000), was recognized by anti-nematode myosin antiserum on immunoblots, and specifically bound to 125I-labelled regulatory and essential light chains in a gel overlay assay. Deletion of 102 residues from the C terminus (mutant 655) reduced regulatory and essential light-chain binding to about 30% and 20% of wild-type levels, respectively. Similar reductions in relative binding of the two light chains were seen with mutant 534, in which 38 residues were deleted from the C terminus. Potential binding sites within 75 residues of the C terminus of S-1 were mapped by construction of five other mutant S-1 clones (398, 399, 400, 409 and 411) containing internal deletions of ten to 12 amino acid residues. These showed up to 30% reductions in their ability to bind essential light chains, but did not differ significantly from wild-type in their ability to bind regulatory light chains. Another mutant, 415, containing a deletion of a conserved acidic hexapeptide, E-D-I-R-D-E, showed enhancement of binding of regulatory and essential light chains to 150% and 165% of wild-type levels. Hence, the major binding sites for both light chains are within 38 amino acid residues of the C terminus.

Amino Acid Sequence↗
PubMed1989

Paramyosin gene (unc-15) of Caenorhabditis elegans. Molecular cloning, nucleotide sequence and models for thick filament structure.

Paramyosin is a major structural component of thick filaments isolated from many invertebrate muscles. The Caenorhabditis elegans paramyosin gene (unc-15) was identified by screening with specific antibodies an "exon-expression" library containing lacZ/nematode gene fusions. Short probes recovered from the library were used to identify bacteriophage lambda and cosmid clones that encompass the entire paramyosin (unc-15) gene. From these clones, numerous subclones containing epitopes reacting with anti-paramyosin sera were obtained, providing strong evidence that the initial cloned fragment was, in fact, derived from the structural gene for paramyosin. The complete nucleotide sequence of a 12 x 10(3) base-pair region spanning the gene was obtained. The gene is composed of ten short exons encoding a protein of 866 [corrected] amino acid residues. Paramyosin is highly similar to residues 267 to 1089 of myosin heavy chain rods. For most of its length, paramyosin appears to form an alpha-helical coiled-coil and shows the expected heptad repeat of hydrophobic amino acid residues and the 28-residue repeat of charged amino acids characteristic of myosin heavy chain rods. However, paramyosin differs from myosin in having non-helical extensions at both the N and C termini and an additional "skip" residue that interrupts the 28-residue repeat. The distribution of charges along the length of the paramyosin rod is also significantly different from that of myosin heavy chain rods. Potential charge-mediated interactions between paramyosin rods and between paramyosin and myosin rods were calculated using a model successfully applied previously to the analysis of the myosin rod sequences. Myosin rods aligned in parallel show optimal charge-charge interactions at multiples of 98 residue staggers (i.e. at axial displacements of multiples of 143 A). Paramyosin rods, in contrast, appear to interact optimally at parallel staggers of 493 residues (i.e. at axial displacements of 720 A) but show only weak interaction peaks at 98 or 296 residues. Similar calculations suggest optimal interactions between paramyosin molecules and myosin rods and in their anti-parallel alignments. The implications of these results for the structure of the bare zone and the assembly of nematode thick filaments are discussed.

Animals↗
PubMed1989

Sequence analysis of the complete Caenorhabditis elegans myosin heavy chain gene family.

The sequences of three myosin heavy chain (MHC) genes from Caenorhabditis elegans, myo-1, 2 and 3, are presented. These genes, together with unc-54, comprise the entire nematode sacromeric MHC family. Comparison of nematode MHC sequences and sarcomeric, smooth and non-muscle MHCs from other organisms highlights conserved sequence features of the MHC rod believed to be important for thick filament assembly. These include: conservation of sequence differences between individual 28 amino acid repeats; invariant placements of large aromatic residues, such as tryptophan, in the rod sequences; conservation of "weak spots" in the hydrophobic seam; and conservation of non-uniform charge distributions along the length of the rod. The rod sequences of the body wall isoforms A and B are more closely related to each other than to the pharyngeal isoforms C and D, suggesting that structural constraints have been imposed by their location within the same thick filament. We have also identified the major transcriptional start site for gene unc-54. Surprisingly, there are no TATA or other known transcription factor elements immediately upstream from the unc-54 start site, or in the upstream regions of the other genes of the C. elegans MHC gene family.

Amino Acid Sequence↗
PubMed1989

Human immunodeficiency virus 1 tat protein binds trans-activation-responsive region (TAR) RNA in vitro.

tat, the trans-activator protein for human immunodeficiency virus 1 (HIV-1), has been expressed in Escherichia coli from synthetic genes. Purified tat binds specifically to HIV-1 trans-activation-responsive region (TAR) RNA in gel-retardation, filter-binding, and immunoprecipitation assays. tat does not bind detectably to antisense TAR RNA sequences, cellular mRNA sequences, variant TAR RNA sequences with altered stem-loop structures, or TAR DNA.

Base Sequence↗
PubMed1989

Transformation of growth factor-dependent myeloid stem cells with retroviral vectors carrying c-myc.

Myeloid progenitor cells and macrophages derived from bone marrow and spleen were efficiently transformed in vitro by infection with Moloney-based retroviral vectors carrying a human c-myc gene. Infected cells were plated in agar in the presence of combinations of the murine lymphokines CSF-1, IL-3, GM-CSF and IL-1. Between 20% and 100% of the colony-forming cells in the initial bone marrow or spleen population could be infected and gave rise to drug-resistant colonies. A large fraction of the infected cells showed continued proliferation after transfer to liquid media and we have derived over 200 growth factor-dependent cell lines. These include adherent and non-adherent CSF-1 or GM-CSF dependent macrophages and macrophage precursors and cell lines which require complex combinations of growth factors for optimal growth. Each of the cell lines displays a unique pattern of expression of surface markers specific for the myeloid lineage including the Mac-1, Mac-2, Mac-3, Ser-4 and F4/80 antigens. Surface markers not specifically associated with the myeloid lineage such as the MHC class II antigens and the Fc-receptor; and surface markers normally associated with the B-cell and T-cell lineages such as B220, L3T4 and Thy1.2 are also found on these cell lines.

Animals↗