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R F Weaver

Publications and source records attributed to R F Weaver.

At least 19 recordsLinked to original sources
PubMed2001

Anti-leukemia selectivity in actinomycin analogues.

An excellent anti-leukemia activity has been found in a group of actinomycin D analogues derivatized at the 2,2'- or 5,5'-position of the depsipeptides. On the basis of the water solubilities, the DNA binding affinities, the RNA synthesis inhibitory activities, the anticancer activities of actinomycin D (AMD), and the crystal structures of DNA-AMD complexes, it becomes clear that AMD is extremely well designed as an effective poison produced by micro-organisms. The anticancer activity of AMD is mainly due to its selective inhibition of RNA synthesis. We have hypothesized that a modification on the AMD structure at a site not involved in DNA interaction can either increase or decrease the diffusion rate of the analogue into certain cancer cells. Since the i-propyl groups of the D-valine residues at the 2,2'-positions and N-methyl-L-valine residues at the 5,5'-positions in the depsipeptides do not participate in interaction with DNA, these amino acid residues were replaced with other D-amino acid residues and N-methyl-L-amino acid residues, respectively. The cancer screen tests have indicated that AMD analogues 2,2'-D-PheAMD, 2,2'-D-OmeAMD, 5,5'-L-TyrAMD, 5,5'-D-ValAMD, 5,5'-D-TyrAMD, 5,5'-D-PheAMD, and 5,5'-D-OmeAMD, inhibit selectively the growth of leukemia cell lines at about 100- to 500-fold lower drug concentrations than those required to inhibit other cancer cell lines.

Animals↗
PubMed1999

Nonessential role for methionines in the productive association between calmodulin and the plasma membrane Ca-ATPase.

To investigate the role of hydrophobic interactions involving methionine side chains in facilitating the productive association between calmodulin (CaM) and the plasma membrane (PM) Ca-ATPase, we have substituted the polar amino acid Gln for Met at multiple positions in both the amino- and carboxyl-terminal domains of CaM. Conformationally sensitive fluorescence signals indicate that these mutations have little effect on the backbone fold of the carboxyl-terminal domain of CaM. The insertion of multiple Gln in either globular domain results in a decrease in the apparent affinity of CaM for the PM-Ca-ATPase. However, despite the multiple substitution of Gln for four methionines at positions 36, 51, 71, and 72 in the amino-terminal domain or for three methionines at positions 124, 144, and 145 in the carboxyl-terminal domain, these mutant CaMs are able to fully activate the PM-Ca-ATPase. Thus, although these CaM mutants have a decreased affinity for the CaM-binding site on the Ca-ATPase, they retain the ability to fully activate the Ca-ATPase at saturating concentrations of CaM. The role of individual methionines in modulating the affinity between the carboxyl terminus and the PM-Ca-ATPase was further investigated through the substitution of individual Met with Gln. Upon substitution of Met(124) and Met(144) with Gln, there is a 5- and 10-fold increase in the amount of CaM necessary to obtain half-maximal activation of the PM-Ca-ATPase, indicating that these methionine side chains participate in the high-affinity association between CaM and the PM-Ca-ATPase. However, substitution of Gln for Met(145) results in no change in the apparent affinity between CaM and the PM-Ca-ATPase, indicating that in contrast to all other known CaM targets, Met(145) does not participate in the interaction between CaM and the PM-Ca-ATPase. These results emphasize differences in the binding interactions between individual methionines in CaM and different target enzymes, and suggest that hydrophobic interactions between methionines in CaM and the binding site on the PM-Ca-ATPase are not necessary for enzyme activation. Calculation of the binding affinities of individual CaM domains associated with activation of the PM-Ca-ATPase suggests that mutations of methionines located in either domain of CaM can decrease the initial high-affinity association between CaM and the PM-Ca-ATPase, but have little effect upon the subsequent binding of the opposing globular domain. These results suggest that the initial associations between CaM and the CaM-binding sequence in the PM-Ca-ATPase are guided by nonspecific hydrophobic interactions involving both domains of CaM.

Amino Acid Substitution↗
PubMed1998

Identification and characterization of a protein kinase-interacting protein encoded by the Autographa californica nuclear polyhedrosis virus.

We have used a yeast two-hybrid system to identify a baculoviral gene encoding a protein kinase-interacting protein (PKIP). The pkip gene is located at 15.8-16.2 m.u (map units) and represents ORF 24 in the sequence of Ayres et al. (1994) of the Autographa californica nuclear polyhedrosis virus. PKIP is a 19.2-kDa protein and appears to stimulate the activity of the viral protein kinase-1 in vitro. Northern blotting analysis revealed that pkip is a late gene. Attempts to plaque purify a pkip- virus were unsuccessful.

Amino Acid Sequence↗
PubMed1998

Overexpression of the 77-kD echinoderm microtubule-associated protein (EMAP), a WD-40 repeat protein, in baculovirus-infected Sf9 cells.

The purpose of this study was to test whether any assembly-promoting microtubule-associated protein (MAP) would bundle microtubules and induce process formation in recombinant baculovirus-infected Sf9 cells, in particular, whether a non-neural MAP from a normally rounded cell would produce cellular asymmetries. To carry out these experiments, we constructed a recombinant baculovirus that expressed the full-length 77-kD EMAP, an abundant MAP that localizes to the mitotic spindle of cleavage-stage sea urchin embryos and to the interphase array of microtubules in adult coelomocytes. Expression of EMAP in Sf9 cells had no detectable effect on cellular morphology, microtubule organization, or stability. These results indicate that process formation in Sf9 cells is MAP specific.

Animals↗
PubMed1997

Selectivity of F8-actinomycin D for RNA:DNA hybrids and its anti-leukemia activity.

Although many compounds have been found that bind to DNA in various ways and exhibit various biological activities, few compounds that specifically bind to RNA or RNA:DNA hybrids are known, even though such compounds are expected to have important biological properties. For example, one characteristic function of the retroviruses, which is generally not found in eukaryotic cells, is the production of an RNA:DNA hybrid in the viral replication phase. If an agent is designed to bind only to an RNA:DNA hybrid, and not to DNA or to RNA, such an agent might be able to inhibit specifically the RNase H activity of retroviral reverse transcriptase, and therefore suppress viral replication. Actinomycin D is known to bind to double-stranded DNA, but not to RNA, because steric hindrance between the 2-amino group of the phenoxazone ring and the 2'-hydroxyl group of RNA prevents intercalation of the compound. However, if the > C-H moiety at the 8-position of the phenoxazone ring is replaced by a > C-F, a possible hydrogen-bond acceptor, this analogue (8-fluoro-actinomycin D, F8AMD) might be able to bind intercalatively to an RNA:DNA hybrid by forming an additional hydrogen bond between F8 and the 2'-hydroxyl group of the guanosine ribose. To test this hypothesis, the crystal structure of d(GAAGCTTC)2-F8AMD has been determined at 3.0 A resolution. Based on this crystal structure, a model in which F8AMD binds into the hybrid r(GAAGCUUC):d(GAAGCTTC) has been built using molecular mechanics and dynamic methods. These structural studies indicate that F8AMD binds intercalatively to a B-form double-stranded DNA whereas the drug intercalates into an RNA:DNA hybrid taking an A-form conformation. In the RNA:DNA hybrid complex, the F8 atom is located so as to be able to interact to an O2' hydroxyl group with either an O-H...F hydrogen bond or H+...F- electrostatic interaction. This interaction might stabilize the F8AMD molecule in the RNA:DNA hybrid. A binding study indicates that both actinomycin D (AMD) and F8AMD bind intercalatively not only to double-stranded DNAs, but also to RNA:DNA hybrids. Although the overall binding capacity of F8AMD (k = 4.5 x 10(5) M-1) is reduced slightly in comparison with AMD itself (k = 1.8 x 10(6) M-1), F8AMD tends to bind relatively more favorably than AMD to the RNA:DNA hybrids. The drugs' effects on RNA synthesis in HeLa cells indicates that the binding capacities of AMD and F8AMD correlates strongly to their RNA synthesis inhibitory activities. F8AMD required a concentration of 78 nM to inhibit RNA polymerase activity in HeLa cells by 50%, whereas AMD reached the same inhibitory level at 30 nM. Surprisingly, F8AMD exhibits unique selectivity against leukemia cells as does another C8-derivatized AMD analogue, N8AMD. F8AMD inhibits 50% of leukemia cell growth at less than 1.0 nM whereas 10- to 130-fold-higher drug concentrations are required to inhibit the growth of other tumor cell lines by 50%. The GI50 value of F8AMD for leukemia cells is the lowest among the GI50 values for all other AMD derivatives tested. By contrast, AMD is quite potent and kills most cells at less than 50 nM concentration, but it does not show any selectivity for certain cell lines. This indicates that AMD should have very limited use as an antitumor agent. It is difficult to rationalize why F8AMD and N8AMD show such strong selectivity against leukemia cells. However, this study and our previous study (J. Am. Chem. Soc. 1994, 116, 7971) indicated that F8AMD and N8AMD tended to bind more favorably to RNA:DNA hybrids. Thus, the unique antileukemia selectivity shown by F8AMD and N8AMD might be used by the agents binding to RNA:DNA hybrids rather than to double-stranded DNA.

Antineoplastic Agents↗
PubMed1996

Physical and biological characteristics of the antitumor drug actinomycin D analogues derivatized at N-methyl-L-valine residues.

The crystal structure of the DNA-actinomycin D (AMD) complex and a simple molecular modeling study indicated that AMD analogues derivatized at N-methyl-L-valine residues (fifth amino acid residue in the cyclic depsipeptide of AMD) could bind to DNA as strongly as the parent AMD. The analogues in which N-methyl-L-valine residues were replaced with L- and D-forms of N-methylvalines, N-methylthreonines, N-methylphenylalanies, N-methyltyrosines, and N-methyl-O-methyltyrosines have been totally synthesized. The characteristics of binding of the analogues to various DNAs including DNA-1 [d(TATATATGCATATATA)], DNA-2 [d(TATATACGCGTATATA)], DNA-3 [d(ATATATAGCTATATAT)], and DNA-4 [d(ATATATGGCCATATAT)] have been examined by using visible absorption spectrum methods. The association constants calculated from the absorption spectra indicate that the modifications of the N-methyl-L-valine residues in the AMD molecule do affect the DNA binding characteristics of the analogues. The L-aromatic analogues bind slightly better than the L-aliphatic analogues except for binding to DNA-1 (-TGCA-), whereas the D-aliphatic analogues bind consistently better than the D-aromatic analogues. In the L-form analogues, the L-Tyr analogue has the highest overall association constant, whereas the D-Val analogue has the highest association constant among the D-form analogues. In spite of substitution of bulky aromatic groups, the D-aromatic analogues bind to the DNA-1 quite well. However, D-aromatic analogues have significantly reduced their binding capacities to the other DNAs, indicating that the substitution of the D-aromatic residues creates a unique four-base sequence preference (-TGCA-). The RNA polymerase inhibitory activities of the AMD analogues in vivo have been examined using human cells (HeLa). All AMD analogues except for the L-Thr analogues severely inhibit RNA synthesis at relatively low drug concentrations. The D-Val, L-OMT, L-Phe, and D-Phe analogues inhibit RNA synthesis more strongly than the natural antibiotic (AMD itself).

Antibiotics, Antineoplastic↗
PubMed1996

Temperature-sensitive mutations in the protein kinase-1 (pk-1) gene of the Autographa californica nuclear polyhedrosis virus that block very late gene expression.

We have developed a genetic screen for temperature-sensitive mutations in the very late transcription apparatus of the Autographa californica nuclear polyhedrosis virus. This method starts with the BacPAKS virus, which has the Escherichia coli lacZ gene under the control of the very late polyhedrin promoter. The desired mutants are temperature-sensitive for beta-galactosidase production and can be complemented by wild-type virus, which lacks the lacZ gene. Two mutants created by nitrosoguanidine mutagenesis and identified by this screen, and one mutant identified by another screen, have been mapped by marker rescue to the viral protein kinase 1 gene (pk-1). The protein kinase genes of these three mutant viruses have been sequenced, revealing the same point mutation in two of them and a different point mutation in the other. In each case, a single amino acid is changed: In two mutants, XF4 and XF5, Asp 92 is changed to Asn; in the other mutant, KT800, Thr 204 is changed to lle. Northern blotting of RNA made in cells infected by these three mutant viruses has shown that the accumulation of very late transcripts (lacZ and p10) is temperature-sensitive, but that accumulation of at least one late transcript (vp39) is not temperature-sensitive. Nuclear run-on transcription assays with two of the mutants indicate that very late transcription is somewhat temperature-sensitive, although this defect is not as pronounced as the temperature-sensitivity detected by Northern blotting. Transcription of at least one late gene (vp39) is not temperature-sensitive in cells infected by these two mutants. Thus, it appears that the viral protein kinase-1 is involved in very late gene expression. Some of this effect is at the transcription level, but some may also be exerted at the posttranscription level.

Amino Acid Sequence↗
PubMed1996

Purification of a virus-induced RNA polymerase from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells that accurately initiates late and very late transcription in vitro.

The virus-induced RNA polymerase from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells was separated from the three host nuclear RNA polymerases by DEAE-Sephadex chromatography and then purified through two more steps: heparin-agarose chromatography and glycerol gradient ultracentrifugation. Fractions from each of these purification steps have been assayed in vitro for the ability to perform accurate initiation of transcription on a late (p6.9) and a very late (polyhedrin) template using primer extension analysis. In each case, the ability to accurately initiate transcription of these genes coincided with the virus-induced polymerase activity. Only after the glycerol gradient ultracentrifugation step did significant amounts of nonspecific late initiation occur, but specific late initiation was still readily detectable, suggesting that there is a limited number of late transcription factors, or that the factors are stably bound in a complex. After the glycerol gradient ultracentrifugation step, SDS-PAGE showed fewer than 10 prominent polypeptides remaining in the active fractions, which suggests a high degree of purity of the transcription machinery.

Animals↗
PubMed1996

Purification of porcine phospholamban expressed in Escherichia coli.

Phospholamban (PLB) is a small hydrophobic protein that regulates contractility in the heart. This membrane protein expressed in bacterial cells is resistant to purification by conventional strategies that have been used to isolate expressed soluble proteins. Therefore, in order to obtain both wild-type and mutant PLB proteins, we have amplified the PLB gene by the polymerase chain reaction from genomic DNA of porcine heart and inserted it into the pGEX-2T plasmid expression vector. In this vector, the gene product fused to glutathione S-transferase has been expressed in JM109 Escherichia coli cells. The expressed fusion protein was found associated predominantly with insoluble cellular constituents. However, most of the fusion protein was readily extracted with SDS. PLB was subsequently purified by a simple procedure consisting of isolation of the fusion protein by preparative SDS-gel electrophoresis, followed by a second electrophoretic separation of PLB after its cleavage from the fusion protein by thrombin. This isolation method yields 3-4 mg of PLB per liter of cells, in a form which is capable of functional interaction with the Ca-ATPase in reconstituted proteoliposomes.

Animals↗
PubMed1995

Structural, physical, and biological characteristics of RNA.DNA binding agent N8-actinomycin D.

The crystal structure of the self-complementary DNA octamer d(GAAGCTTC)2 complexed with N8-actinomycin D (N8AMD) has been determined at 3.0 A resolution (space group: P3(1)21; unit cell: a = 62.30, b = 62.30, c = 42.97 A; R = 0.173 for 1845 reflections). The DNA structure was severely distorted by the N8AMD bound intercalatively into the middle dinucleotide, 5'-GC-3'. The two cyclic depsipeptides, which differ from each other in overall conformation, lie in the minor groove. The complex is further stabilized by forming base--peptide and chromophore--backbone hydrogen bonds. The complexes are stacked together to form a pseudocontinuous helix running through the crystals. The structure of d(GAAGCTTC)2-actinomycin D (AMD) crystallized in the space group C2 [Kamitori S., & Takusagawa, F. (1992) J. Mol. Biol. 225, 445-456] was re-refined in order to compare it directly to the N8AMD complex structure. The asymmetrical binding mode of AMD has been confirmed on the basis of the two complex structures. The crystal structures of the N8AMD and AMD complexes bound to the same d(GAAGCTTC)2 differed by a root-mean-square deviation on all atom positions of 1.77 A, but most of the structural differences can be attributed to molecular packing in two different crystal forms, and not to structural differences induced by the interaction with the intercalating agents. However, the DNA binding and biological characteristics of N8AMD and AMD are quite different from each other. The DNA association constant of N8AMD is 33-fold less than that of AMD in an aqueous solution. N8AMD required a concentration > 10.0 microM to inhibit RNA synthesis activity in HeLa cells by 50%, whereas AMD reached to the same inhibitory level at only 35 nM. The structure of the DNA-N8AMD complex suggested that substitution of the N-methyl-L-valine residue in the cyclic depsipeptide with a N-methyl-D-valine residue might increase the hydrophobic interaction with the minor groove of the DNA. Thus the DNA association constant and RNA synthesis inhibitory activities of 5,5'-N-methyl-D-valine AMD (D-MeVal-AMD) have also been determined. The DNA association constant of D-MeVal-AMD is more than 2-fold greater than that of AMD, and the RNA synthesis inhibitory activity is about 20-fold greater.

Base Sequence↗
PubMed1993

Transcription mapping and functional analysis of the protein tyrosine/serine phosphatase (PTPase) gene of the Autographa californica nuclear polyhedrosis virus.

The protein tyrosine/serine phosphatase (PTPase) gene of the Autographa californica nuclear polyhedrosis virus yields two major transcripts of approximate sizes 3.1 and 3.9 kb. Both of these are very late transcripts, which accumulate to maximal levels more than 30 hr postinfection. The smaller transcript initiates at the T of an ATAAG sequence that lies 22 bp upstream of the putative start codon of the gene. The larger transcript initiates at the first A of a TTAAG sequence that lies approximately 798 bp further upstream. Thus, the larger transcript is made by transcribing through the entire hr1 region, which lies just 60 bp upstream of the putative translation start site. We have expressed the product of this gene as a fusion protein with glutathione-S-transferase and have shown that it has PTPase activity similar to that of the vaccinia virus H1 gene product: It dephosphorylates both protein phosphotyrosines and phosphoserines/phosphothreonines, and it is inhibited by vanadate, but not by okadaic acid.

Amino Acid Sequence↗
PubMed1991

Structural comparison of the Autographa californica nuclear polyhedrosis virus-induced RNA polymerase and the three nuclear RNA polymerases from the host, Spodoptera frugiperda.

A partial subunit structure has been determined for the novel RNA polymerase that is induced in fall armyworm (Spodoptera frugiperda) cells upon infection with the Autographa californica nuclear polyhedrosis virus (AcNPV). The putative structure includes nine polypeptides; the complexity of this structure is in accord with the high sedimentation coefficient (15S) estimated for this enzyme. A comparison of the putative structure of the virus-induced polymerase with those of the three host nuclear RNA polymerases shows that the structure of the viral polymerase is apparently unlike any of the host nuclear polymerases. This conclusion is reinforced by immunoblot experiments that show no cross-reactivity between the virus-induced polymerase and an antiserum directed against Drosophila RNA polymerase II. The virus-induced RNA polymerase appears at the onset of the late phase of infection and still appears when viral DNA synthesis is blocked by aphidicolin. Thus, the virus-induced polymerase seems to be composed of early viral products.

Animals↗
PubMed1991

Nucleotide sequence and transcript mapping of the HindIII F region of the Autographa californica nuclear polyhedrosis virus genome.

The organization of genes in the 2.95 kb EcoRI-SalI fragment (0.7 to 3.0 map units) located within the HindIII F region of the Autographa californica nuclear polyhedrosis virus genome was studied by a combination of DNA sequencing, Northern blot analysis, S1 mapping and primer extension analysis. In addition to the two divergent overlapping transcripts [leftward early (ES1) and rightward late (ES2)] previously reported, a third transcript which is present at 18 h p.i. and which also runs leftward, overlapping ES1 by 1600 nucleotides (nt) at the 3' end, was mapped to this region. The DNA sequence revealed the presence of three open reading frames (ORFs) of significant length. ORF-1 and ORF-2 correspond to the leftward transcripts, and code for potential polypeptides of 151 and 329 amino acids, respectively. ORF-3 which codes for a potential polypeptide of 167 amino acids is located on the opposite strand in a region for which no transcript mapping data are available. However, the conserved late gene promoter/cap site sequence (ATAAG) is present 23 nt upstream of the start of ORF-3.

Amino Acid Sequence↗
PubMed1990

Identifying the RNA polymerases that synthesize specific transcripts of the Autographa californica nuclear polyhedrosis virus.

Nuclear run-on assays carried out in the presence and absence of the RNA polymerase II inhibitor, alpha-amanitin, were used to determine the exact timing of the switch from inhibitor-sensitive transcription catalysed by host RNA polymerase II, to inhibitor-resistant transcription catalysed by the baculovirus-induced RNA polymerase. These studies revealed that the onset of alpha-amanitin-resistant transcription is just after 6 h post-infection, simultaneous with the beginning of the late phase of infection. They also showed that transcripts from the p26 gene in the HindIII Q/P region and the p35 gene in the HindIII K/Q region of the viral genome are synthesized by the host RNA polymerase II both early and late in infection. On the other hand, transcripts of the p10 gene in the HindIII Q/P region and the gamma transcripts in the HindIII K region are synthesized by the alpha-amanitin-resistant, virus-induced RNA polymerase late in infection.

Amanitins↗
PubMed1990

Overlapping divergent transcripts mapping to the HindIII F region of the Autographa californica nuclear polyhedrosis virus.

We have mapped a set of transcripts that cross the XhoI site in the HindIII F fragment of the genome of the Autographa californica nuclear polyhedrosis virus. These transcripts overlap at their 5' ends by about 550 bases and run in opposite directions. We have tentatively identified two open reading frames corresponding to the leftward transcript. The rightward transcript is present from 8 h post-infection (p.i.) to 24 h p.i.; the leftward transcripts are present from 2 h p.i. to 24 h p.i. The early (2 h) transcript is about 2.1 kb in size and its 5' end maps about 504 bp to the right of the XhoI site. Beginning at about 8 h p.i. a new transcription start site is used, about 80 bp downstream (to the left) of the first. The late (rightward) transcript is about 1.2 kb in size; its 5' end seems to be heterogeneous and maps about 44 to 60 bp to the left of the XhoI site. Late in infection transcription proceeds in both directions at the same time through the overlapping region of the DNA encoding these transcripts.

Animals↗
PubMed1990

Categorizing some early and late transcripts directed by the Autographa californica nuclear polyhedrosis virus.

Using an S1 mapping assay on RNA from Spodoptera frugiperda cells infected by the Autographa californica nuclear polyhedrosis virus in the presence and absence of cycloheximide and aphidicolin, we can distinguish three classes of transcripts. First, there are those whose synthesis is blocked by the DNA synthesis inhibitor aphidicolin and which are therefore late transcripts. These include the late transcript of the 39K gene and a late leftward transcript across the XhoI site in the HindIII-F region. Second, there are those whose synthesis is not blocked by aphidicolin, but whose accumulation is inhibited by the protein synthesis inhibitor cycloheximide and which are therefore presumably delayed early genes. These include the p26 transcript(s), the early 39K transcript and the alpha 2 transcript in the HindIII-K/Q region. Third, there are those whose accumulation is not affected or is enhanced by cycloheximide. These are not necessarily immediate early transcripts, but their response to cycloheximide is clearly different from that of those in the second class. They include the alpha 1 and alpha 3 transcripts in the HindIII-K/Q region and the early leftward transcript across the XhoI site in the HindIII-F region.

Animals↗
PubMed1989

Studies on the control region of the p10 gene of the Autographa californica nuclear polyhedrosis virus.

5' deletion mutants of the Autographa californica nuclear polyhedrosis virus very late p10 gene promoter have been prepared and subjected to a transient expression assay in infected Spodoptera frugiperda cells. The control plasmid contained the chloramphenicol acetyltransferase (CAT) reporter gene under the control of the p10 promoter, which was included in a 230 bp sequence upstream from the p10 translation initiation codon. The control plasmid also contained a segment of the hr5 enhancer downstream from the CAT gene. Promoter activity was unaffected by 5' deletion to position -77, which lies about 11 bp upstream from the p10 cap site. However, deletion of 12 more bp completely eliminated p10 promoter activity. Thus, the 5' border of the p10 promoter lies downstream from position -77, and the region between positions -77 and -65 contains an element that is important to promoter activity. This is the region that is conserved near the cap sites of late baculovirus genes. Our studies also show that transient expression of CAT under the control of the p10 promoter and hr5 enhancer is higher when transfection occurs prior to infection by virus.

Animals↗