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B Amit

Publications and source records attributed to B Amit.

9 recordsLinked to original sources
PubMed1996

Molecular analysis of the second template switch during reverse transcription of the HIV RNA template.

The molecular events leading to the second template switch during reverse transcription of the HIV genome were studied in a defined in-vitro system. In order to investigate displacement of the tRNA(lys) primer from the primer binding site (PBS) of the viral genomic RNA, following DNA synthesis, we produced an HIV RNA/DNA substrate that resembles the intermediate reverse transcription complex formed prior to the second template switch. Partial tRNA(lys) primer displacement was observed during plus (+) strand DNA synthesis and during minus (-) strand DNA elongation. We found two determinants that may serve as a stop signal for (+) DNA strong stop synthesis, the A(m) at position 19 of the natural tRNA(lys) and the secondary structure at the PBS sequence. The later signal appears to constitute a stronger terminator in-vitro. The 3' end of the nascent (-) DNA strand prior to the second template switch was also determined. It was mapped to the U5-PBS junction at the site for the first endonucleolytic cut introduced by the RNase H activity of the HIV reverse transcriptase (RT). Thus, different signals dictate the arrest of (-) and (+) nascent DNA synthesis. These stop signals appear to be required for the subsequent second template switch. However, an excess of (-) DNA "acceptor" molecules, having a 18-base sequence complementary to the (+) DNA "donor" template, was required to demonstrate the actual template switch in the in-vitro system. Taken together these results indicate that the reverse transcriptase can catalyze all the steps leading to the second template switch and auxiliary viral proteins may act to enhance the efficiency of this step during the reverse transcription process.

Base Sequence↗
PubMed1996

Ribonuclease H activity during initiation of reverse transcription using tRNA(lys)/RNA primer/template of human immunodeficiency virus.

The specificity of the initial cleavage by the RNaseH activity of HIV-1 reverse transcriptase (RT) during minus strong-stop DNA synthesis was studied using the authentic primer/template tRNA(lys)/HIV RNA. We observed that concomitant with the initiation of DNA synthesis, RNaseH activity of HIV RT introduced the first endonucleolytic cuts within the U5 region of the HIV RNA template, mainly 1 and 3 bases away from the primer binding site. To analyze whether the cleavage sites were determined by sequence specificity, the authentic U5 region at one of the cleavage sites was mutated. The change of sequence did not alter the initial cleavage pattern of RNaseH. In order to determine the size of the RNA/DNA hybrid that is required for RNaseH activation during reverse transcription initiation, DNA synthesis was limited by dideoxynucleotides. DNA extension of the tRNA(lys) primer by 17 deoxyribonucleotides but not by 6 deoxyribonucleotides was sufficient to activate the RNaseH site of HIV RT. Taken together, our results indicate that during initiation of minus strongstop DNA synthesis by HIV RT, the first RNaseH-mediated endonucleolytic cut of the genomic RNA is dictated mainly by the length of the nascent DNA and not by sequence preference.

Base Sequence↗
PubMed1994

Cellular distribution of HIV type 1 Nef protein: identification of domains in Nef required for association with membrane and detergent-insoluble cellular matrix.

Cellular distribution of HIV-1 Nef protein was studied by expressing the protein in mammalian cells. Cell extracts were fractionated by low- and high-speed centrifugation and by nonionic detergents. Two Nef-related proteins were expressed in COS cells, Nef-27kD and Nef-25kD. Nef-27kD, an N-myristoylated form of Nef, was found in the cytosol and in association with a particulate fraction of the cytoplasm. Treatment of the particulate cytoplasmic fraction with nonionic detergents, using three different protocols designed to isolate the cytoskeleton matrix, indicated that part of Nef was sensitive and part was resistant to detergent solubilization. These two cellular fractions represent membrane- and cytoskeleton-associated Nef. Nef-25kD, initiated from an in-frame AUG codon, was not modified with myristic acid at the amino terminus. Consequently, this protein was present in a soluble form in the cytosol. Furthermore, a mutant of Nef-27kD, in which the myristoylation signal is deleted, appears as a cytoplasmic soluble protein. To determine domains in Nef that are responsible for its subcellular distribution, successive internal deletions of 14-20 amino acids were introduced at the N-terminal portion of the protein. Five mutants were evaluated with respect to their cellular localization. One mutant (pSVLA-5), from which amino acids 73-88 were deleted, did not copurify with the detergent-insoluble fraction. The protein was, however, present in the particulate cytoplasmic fraction, presumably in association with membranes. Taken together, these results suggest that N-myristoylation of Nef affects its association with both membranes and cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

RNase H activity of reverse transcriptases on substrates derived from the 5' end of retroviral genome.

RNA/DNA substrates derived from the 5' ends of human immunodeficiency virus (HIV) and Moloney murine leukemia virus (MMuLV) genomes were used to study the specificity of the RNase H activities of HIV, AMV (avian myeloblastosis virus), and MMuLV reverse transcriptases. These substrates were selected because they represent the site for the first template switch during proviral DNA synthesis. Variability of cleavage was observed depending on the origin of the enzyme as well as the sequence of the RNA/DNA substrate. The minimal size of hybrid recognized by the RNase H activity of reverse transcriptase was also affected by the same parameters, namely, the enzyme and the substrate origin. Moreover, the size of the residual 5'-undigested RNA after completion of the RNase H reaction depended on the position of the DNA annealed to the genomic RNA. When the hybrid was located at the 5' R region of the viral genome, stable hybrids with RNAs of 13-18 nucleotides remained following digestion by HIV reverse transcriptase, and 21-24 nucleotides following digestion by AMV reverse transcriptase and MMuLV reverse transcriptase. On the other hand, with all three enzymes, smaller sized hybrids remained when the DNA was hybridized to internal U5 or R sequences. The reason for this variance in size appears to be the inability of RNase H to efficiently digest at the 5' end of hybrid structures. Surprisingly, hybridization to the RNA template, of a DNA oligomer that extended 15 nucleotides beyond the 5' end of the RNA R region sequences, resulted in further digestion of the RNA. This unexpected mode of action of RNase H at the 5' end of the genomic RNA should be taken in consideration in studies of the first template switch.

Avian Myeloblastosis Virus↗
PubMed1990

A constitutive expression vector system driven by the deo P1P2 promoters of Escherichia coli.

The P1P2 promoters of Escherichia coli K12 deo operon, residing on an AvaII restriction fragment, were used to construct a new expression vector. To evaluate the potential of the P1P2-driven expression system we have inserted the sequence of human superoxide dismutase (hSOD) downstream of the deo ribosome binding site. Expression of hSOD was evaluated by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and enzyme activity. In crude cell extracts hSOD expression levels were found to be high in hosts possessing no deoR or cytR repressors. Highest levels of hSOD expression were obtained with a high-copy-number plasmid regardless of the host used. Expressed hSOD can account for 35%-40% of total protein in E. coli.

Escherichia coli↗
PubMed1990

Expression and biochemical characterization of human immunodeficiency virus type 1 nef gene product.

nef genes from human immunodeficiency virus type 1 isolates BH10 and LAV1 (lymphadenopathy-associated virus type 1) were expressed in Escherichia coli under the deo operon promoter. The two proteins found in the soluble compartment of the bacterial lysate were purified by ion-exchange column chromatography to apparent homogeneity. Determination of the amino-terminal sequence revealed glycine as the first amino acid in the Nef protein, indicating removal of the initiator methionine during expression in E. coli. Under native conditions, the recombinant Nef protein is a monomer of 23 kilodaltons. In denaturing polyacrylamide gels, however, BH10 and LAV1 Nef proteins migrate as 28 and 26 kilodaltons, respectively. GTP binding and GTPase activity were monitored during Nef protein purification. These activities did not copurify with the recombinant Nef protein from either the BH10 or the LAV1 isolate. Purified recombinant BH10 Nef protein was used as an immunogen to elicit mouse monoclonal antibodies. A series of monoclonal antibodies were obtained which reacted with sequences at either the amino or carboxy terminus of Nef. In addition, a conformational epitope reacting with native BH10, but not LAV1, Nef was isolated.

Amino Acid Sequence↗
PubMed1988

Site-specific mutagenesis of human apolipoprotein E. Receptor binding activity of variants with single amino acid substitutions.

Apolipoprotein (apo) E, an important protein involved in cholesterol transport in the plasma, binds with high specificity and high affinity to the apoB, E (low density lipoprotein) receptor. Several lines of evidence have indicated that key basic residues in the vicinity of residues 140-160 of apoE are important in mediating binding to the receptor. Furthermore, apoE variants exhibiting defective receptor binding are associated with the genetic lipid disorder type III hyperlipoproteinemia. To determine whether other basic amino acids in this region of apoE also affect receptor binding activity, site-specific mutagenesis of apoE in a bacterial expression system was undertaken. This system had been used successfully to produce apoE3 that was structurally and functionally equivalent to human plasma apoE3. Variants of apoE in which neutral amino acids were substituted for basic residues at positions 136, 140, 143, and 150 were produced. The variants all displayed defective binding; their activity ranged from 9 to 52% of normal (a range similar to that seen with naturally occurring variants of human apoE). In addition, to determine whether the conformation of this region is important for receptor binding, we designed variants in which proline was substituted for leucine 144 or alanine 152. Both variants were defective, exhibiting 13 and 27% of normal binding, respectively. In contrast, a double mutant in which arginine was substituted for serine 139 and alanine for leucine 149 displayed slightly enhanced receptor binding activity. These studies confirm that the middle of the apoE molecule is important in receptor binding and indicate that only certain amino acid substitutions in this region interfere with receptor binding activity.

Amino Acid Sequence↗
PubMed1977

A photolabile protecting group for the phenolic hydroxyl function of tyrosine.

The ortho-nitrobenzyl group (ONBzl) was utilized to protect the phenolic functions of the side chain of tyrosine. Optimal conditions for its mild photolyti removal were established. This paper describes the preparation and properties of some (O-ONBzl)-tyrosine derivatives of potential value in the synthesis of tyrosine-containing peptides. Their use in the synthesis of the dipeptide, N-benzyloxycarbonyl-L-tyrosyl-glycine ethyl ester is presented as an example.

Benzyl Compounds↗