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S Litvak

Publications and source records attributed to S Litvak.

At least 55 records · Page 3Linked to original sources
PubMed1996

Changes of enzymes and factors involved in DNA synthesis during wheat embryo germination.

We have previously purified and characterized wheat germ DNA polymerases A and B. To determine the role played by DNA polymerases A and B in DNA replication, we have measured the level of their activities during wheat embryo germination. The level of cellular proteins known to be associated with DNA synthesis such as PCNA and DNA primase were also investigated. The activity of DNA polymerase A gradually increased reaching a maximal level at 12 h after germination. Three days later, only a residual activity was detected. DNA polymerase B showed the same pattern during germination with very similar changes in activity. Our results indicate a striking correlation between maximal activities of DNA polymerase A, DNA polymerase B and optimal levels of DNA synthesis. These results support a replicative role of these enzymes. The activity of wheat DNA primase that copurifies with DNA polymerase A also increases during wheat germination. Taking together all its properties, and in spite of its behaviour with some inhibitors. DNA polymerase A may be considered as the plant counterpart of animal DNA polymerase alpha. Concerning DNA polymerase B we have previously shown that PCNA stimulates its processivity. Besides studying the changes of DNA polymerases A and B and DNA primase we have also studied changes in PCNA during germination. We show that PCNA is present in wheat embryos at a constant relatively high level during the first 24 h of germination. After 48 h, the absence of PCNA is concomitant with an important decrease in DNA polymerase B activity. In this report we confirm the behaviour of DNA polymerase B as a delta-like activity.

Animals↗
PubMed1996

Expression of functional HIV-1 integrase in the yeast Saccharomyces cerevisiae leads to the emergence of a lethal phenotype: potential use for inhibitor screening.

The integrase of the human immunodeficiency virus type 1 (HIV-1) has been expressed in yeast in order to investigate its potential lethal effect mediated by DNA damage. To this end, we have constructed an expression plasmid containing the retroviral integrase gene under the control of the inducible promotor ADH2/GAPDH which is regulated by the glucose concentration of the medium. Haploid yeast strain W303-1A did not appear to be clearly sensitive to HIV-1 integrase expression. However, disruption of the RAD 52 gene, which is involved in the repair of double-strand DNA breaks, strongly increased the deleterious effects of the retroviral enzyme in this yeast strain. The diploid strain constructed with W303-1A and an isogenic strain of the opposite mating type also showed a strong sensitivity to the HIV-1 integrase. Under yeast culture conditions allowing moderate integrase synthesis, the deleterious effect was totally abolished by missense integrase mutations, which are known to abolish HIV-1 integrase activities in vitro. We conclude that the lethal phenotype due to HIV-1 integrase expression in yeast may be closely related to the HIV-1 integration reaction in infected human cells, and that yeast may be a useful tool to study the HIV-1 integration process and to screen drugs capable of inhibiting HIV-1 integration in vivo.

Antiviral Agents↗
PubMed1996

Control of gene expression by base deamination: the case of RNA editing in wheat mitochondria.

The term 'RNA editing' was used for the first time in 1986 to describe the process of uridylate insertion into trypanosomal mitochondrial transcripts. Since then, the term has been used more generally to describe a large variety of processes involving base insertions, deletions and conversions that generate RNAs with a primary sequence different to those encoded by the gene. RNA editing has been observed in the mitochondrial fraction of trypanosomes, plants and other organisms, in the animal nuclear fraction in the case of the apolipoprotein B and glutamate brain receptors mRNAs as well as in viruses like paramyxovirus, hepatitis delta and probably HIV. The role of cytidine and adenine deamination leading to C to U and A to I transitions has became pivotal to explain this process by base conversion. In this review we will focus mainly on the work performed in our group on plant mitochondria and more specifically on the mechanism and the functional significance of RNA editing in wheat organelles. The original contributions of our laboratory in this field are: i) showing that RNA editing is reflected at the protein level; ii) settling three in vitro systems to assay C to U conversion using a wheat mitochondrial lysate as source of enzymes and factors, and unedited mRNA from the same source, as substrate; iii) determination by double labelling of the unedited substrate that RNA editing in wheat mitochondria occurs via a deamination step; and iv) that introducing unedited proteins in the mitochondria of transgenic plants leads to the emergence of cytoplasmic male sterility supporting the idea that the role of this process is to produce functional proteins. Using the antisense approach in transgenic plants we were able to obtain a significant male fertility restoration.

Cytosine Deaminase↗
PubMed1996

Antisense oligonucleotides inhibit in vitro cDNA synthesis by HIV-1 reverse transcriptase.

The inhibition of reverse transcription by various chemically modified antisense oligonucleotides was studied in a cell-free system, composed of an RNA template, a primer oligodeoxynucleotide, and the HIV-1 reverse transcriptase (RT). Different mechanisms of inhibition were observed depending on the chemical structure of the antisense molecule. (1) The hybridization of 2'-O-allyl oligonucleotide to the RNA template promotes a physical arrest of the polymerase. (2) The antisense effect of phosphodiester or phosphorothioate oligonucleotides is essentially due to the RNase H-mediated cleavage of the RNA. (3) A third mechanism was observed with phosphorothioate oligonucleotides that directly interact with the enzyme. Chimeric oligonucleotides, composed of an unmodified region flanked by 2'-O-methyl groups, led to less efficient inhibition than the parent unmodified oligomer, although the inhibitory mechanism was the same. No inhibitory effect was detected when alpha or methylphosphonate oligomers were used.

Antiviral Agents↗
PubMed1996

Consumer-directed models of personal care: lessons from Medicaid.

"Consumer-directed" models of financing and services delivery are compared with models that emphasize professional control and accountability within the context of Medicaid-financed personal care services (PCS). The Medicaid PCS benefit finances aide or attendant services for low-income persons with functional disabilities to assist them with daily living tasks. Consumer-directed modes of service provision permit service recipients themselves to have greater choice and control over all aspects of service provision. Client surveys in three states found that clients were most satisfied with the program elements of Medicaid PCS services that gave them more choice and control. Case studies of how Medicaid PCS programs in particular states are administered indicate that the use of aides who are independent providers, unattached to a home health or home care agency, is a critical aspect of consumer direction. By itself, however, this factor does not guarantee consumer direction because other Medicaid PCS rules and regulations may restrict client choice and control.

Aged↗
PubMed1995

High-affinity interaction of human immunodeficiency virus type-1 reverse transcriptase with partially complementary primers.

The comparison of Km and Vmax values for various primers in the reaction of polymerization catalyzed by the human immunodeficiency virus type-1 (HIV-1) reverse transcriptase was carried out. The primers were: (a) complementary to the template, (b) partially complementary with mismatched nucleotides at different positions from the 3' end or (c) non-complementary. Non-complementary primers were not elongated by HIV-1 reverse transcriptase. However, if they contained only one residue complementary to the template or an abasic unit at the 3' end, they could serve as primers. The most effective discrimination between matched and mismatched primers, due to a decrease in the affinity and Vmax, was found in the case of oligonucleotides containing non-complementary bases at the second or third position from the 3' end of the primer. The efficiency of discrimination by HIV-1 reverse transcriptase between matched and mismatched base-paired primers was about 1-1.5 orders of magnitude lower than that of procaryotic, eucaryotic and archaebacterial DNA polymerases and avian myeloblastosis virus reverse transcriptase. Oligonucleotides such as (dT)4(dCdG)k(dT)4 showed higher affinity for the enzyme than (dT)4 or (dT)8 primers. These data suggest that HIV-1 reverse transcriptase, in contrast to procaryotic, eucaryotic and archaebacterial DNA polymerases, forms additional contacts with the 5'-end region of the non-complementary primer. In addition, using tRNA(3Lys), the natural primer of HIV-1, it was shown that the p66 subunit of reverse transcriptase can be crosslinked, in the presence of a platinum derivative, to the 5' end of tRNA. Thus, besides the normal binding site for the 3' end of tRNA, which is crucial for the initiation of cDNA synthesis, the 5' end of the tRNA also interacts with a specific site on the enzyme.

DNA, Complementary↗
PubMed1995

DNA synthesis primed by mononucleotides (de novo synthesis) catalyzed by HIV-1 reverse transcriptase: tRNA(Lys,3) activation.

HIV-1 RT is able to catalyze DNA synthesis starting from mononucleotides used both as minimal primers and as nucleotide substrates (de novo synthesis) in the presence of a complementary template. The rate of this process is rather slow when compared to the polymerization primed by an oligonucleotide. The addition of tRNA(Lys,3) to this system increased the de novo synthesis rate by 2-fold. Addition of low concentrations of agents able to modify protein conformation, such as urea, dimethylsulfoxide and Triton X-100, can activate the de novo synthesis by a factor 2 to 5. A dramatic synergy is observed in the presence of the three compounds since the stimulating effect of tRNA increases 10-15 times. These results suggest that compounds activating RT are able to induce a conformational change of the enzyme which results in a higher specific activity. Primer tRNA seems to play an important role in HIV-1 RT modification(s) leading to a polymerase having a higher affinity for the primer or the dTTP, but not for the template. The specificity of RT for the template is not influenced by changes in the kinetics or in the thermodynamic parameters of the polymerization reaction.

DNA↗
PubMed1995

RNA editing in wheat mitochondria proceeds by a deamination mechanism.

Most if not all mitochondrial messenger RNAs from seed plants undergo a post-transcriptional modification (RNA editing) involving the conversion of some cytidine residues to uridine. Using a molecular hybridization approach, an in vitro RNA editing system, able to faithfully reproduce the in vivo observed C to U changes of subunit 9 (atp9) of wheat mitochondrial ATP synthase mRNA, has been described [Araya et al. (1992) Proc. Natl. Acad. Sci. USA 89, 1040-1044]. In this work we extend these studies to better understand the biochemical mechanism of this process. RNA editing was analysed by P1 nuclease digestion of the reaction product followed by thin layer chromatography. Experiments performed with unedited [3H]RNA labelled on the base and with unedited [32P]RNA labelled at the alpha-phosphate of cytidine residues, indicate that plant mitochondrial RNA editing operates through a deamination mechanism.

Base Sequence↗
PubMed1995

Sequence-specific inhibition of human immunodeficiency virus (HIV) reverse transcription by antisense oligonucleotides: comparative study in cell-free assays and in HIV-infected cells.

We have investigated two regions of the viral RNA of human immunodeficiency virus type 1 (HIV-1) as potential targets for antisense oligonucleotides. An oligodeoxynucleotide targeted to the U5 region of the viral genome was shown to block the elongation of cDNA synthesized by HIV-1 reverse transcriptase in vitro. This arrest of reverse transcription was independent of the presence of RNase H activity associated with the reverse transcriptase enzyme. A second oligodeoxynucleotide targeted to a site adjacent to the primer binding site inhibited reverse transcription in an RNase H-dependent manner. These two oligonucleotides were covalently linked to a poly(L-lysine) carrier and tested for their ability to inhibit HIV-1 infection in cell cultures. Both oligonucleotides inhibited virus production in a sequence- and dose-dependent manner. PCR analysis showed that they inhibited proviral DNA synthesis in infected cells. In contrast, an antisense oligonucleotide targeted to the tat sequence did not inhibit proviral DNA synthesis but inhibited viral production at a later step of virus development. These experiments show that antisense oligonucleotides targeted to two regions of HIV-1 viral RNA can inhibit the first step of viral infection--i.e., reverse transcription--and prevent the synthesis of proviral DNA in cell cultures.

Base Sequence↗
PubMed1995

Interaction of primer tRNA(Lys3) with the p51 subunit of human immunodeficiency virus type 1 reverse transcriptase: a possible role in enzyme activation.

In the interaction between HIV-1 RT and tRNA(Lys3) each subunit of the heterodimer interacts with tRNA showing a different affinity: Kd (p66) = 23 nM, Kd (p51) = 140 nM. Preincubation of heterodimeric RT with tRNA, at concentrations similar to that of the Kd value for p51, leads to an increase of the catalytic activity on poly(A)-oligo(dT). These results were compared to those using different tRNA analogs: oxidized tRNA, tRNAs lacking one, two or three nucleotides from the 3'-end, or ribo- and deoxyribonucleotides mimicking the anticodon loop sequence. In all cases, tRNA analogs were weaker activators of HIV-1 RT than natural tRNA. A possible mechanism of RT p66/p51 activation by tRNA and its analogs, mediated through the p51 subunit, is discussed.

Anticodon↗
PubMed1995

RNA editing in wheat mitochondria.

C to U transitions in plant mitochondrial mRNA (RNA editing) lead to amino acid changes as well as to the creation of new initiation or termination codons. We established an in vitro system to assay and to dissect the process of wheat mitochondrial mRNA editing. A deamination mechanism explains most easily the observed C to U transitions. Several fractions of organellar protein participate in the editing machinery. Some of these proteins presumably carry the catalytic activity while others are typical RNA binding proteins and may confer specificity to the 'editosome' complex. To investigate the functional properties of protein products synthesized from unedited mRNAs, we constructed transgenic tobacco plants carrying an unedited gene coding for subunit 9 (ATP9) of the ATP synthase complex. The nuclear encoded 'unedited' protein product is targeted to the mitochondria with a heterologous presequence. A significant number of male sterile tobacco plants were obtained suggesting that at least the functional ATP9 protein requires RNA editing. This result suggests a novel approach to obtain artificial male sterile plants by using a physiological effect resulting in CMS which mimics the situation found in many natural populations.

Codon, Initiator↗
PubMed1994

Priming of HIV replication by tRNA(Lys3): role of reverse transcriptase.

The fundamental role played by reverse transcriptase in the replication of retroviruses has stimulated the study of the mechanism of action of this enzyme. The reverse transcriptase of the type 1 human immunodeficiency virus forms a stable complex with its cognate transfer RNA replication primer (tRNA(Lys3)). Here, we outline the role of this enzyme in the selection of its primer tRNA, the annealing of primer tRNA to the complementary region of the retroviral genome, and the first attempts to use the reverse-transcriptase-tRNA complex as a new target for antiviral agents.

Base Sequence↗
PubMed1994

The reverse transcriptase of HIV-1: from enzymology to therapeutic intervention.

The human immunodeficiency virus type 1 (HIV-1) is the etiologic agent of AIDS. Replication of this virus requires the activity of a retrovirus encoded RNA-dependent DNA polymerase, or reverse transcriptase (RT). HIV-1 RT is required for the synthesis of the double-stranded proviral DNA from the single-stranded retroviral RNA genome. HIV-1 RT has two subunits of 66 kDa and 51 kDa. The 66-kDa subunit contains the DNA polymerase and RNase H domains whereas the 51-kDa subunit, obtained by proteolytic maturation of the former subunit, has only the DNA synthetic activity. Two recently reported crystal structures of HIV-1 RT have revealed the very asymmetric structure of this molecule. In addition to providing information concerning the mechanism of nucleic acid polymerization, biochemical and biophysical studies of this enzyme are providing key insights for the design of selective antiviral agents. The multiple activities displayed by reverse transcriptase in the replication of the retroviral genome ensure that this enzyme will remain at the forefront of antiviral strategies in the fight against AIDS and other retrovirus-related pathologies.

HIV Reverse Transcriptase↗
PubMed1993

Human immunodeficiency virus type-1 reverse transcriptase copies very short templates: kinetic and crosslinking analysis.

We describe in this article some properties concerning the cDNA elongation activity of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT). The kinetic parameters of the polymerization reaction catalyzed by HIV-1 RT, using short templates, were studied. Values of Km and Vmax were measured as a function of the oligoadenylate template length: the logarithm of Km increased linearly, with an incremental factor of 2.2, when the template length differs by one nucleotide. Using short templates, olig(A)n (n = 7-14) and primers shorter or longer than the template, HIV-1 reverse transcriptase was able to synthesize polymer products longer than 200 nucleotides. We showed that an oligonucleotide as short as (pA)3 was long enough to serve as template for cDNA synthesis by RT. In the binding of RT to template of different lengths (5 to 14 nucleotides long), two constants were determined differing in each case by a factor of about 10. The three recombinant forms of HIV-1 RT (p66/p51, p66/p66 and p51/p51) were crosslinked to a short template, (pA)14, in the presence of cis-aquahydroxydiamminoplatinum. The efficiency of crosslink of [32P](pA)14 template with each of the subunits of RT correlated well with the affinity of this template to the different forms of RT. In the case of p66/p51, the crosslink occurred mainly with the p66 subunit. These results confirm the important catalytic role of the p66 subunit in the heterodimeric human retroviral polymerase.

Cisplatin↗
PubMed1993

Affinity labeling and functional analysis of the primer binding domain of HIV-1 reverse transcriptase.

Six affinity reagents containing chemically reactive groups, either on the phosphate residue at the 5'-end or on the 5'- or 3'-end internucleoside phosphate linkages of the oligothymidylate primers, were used to covalently modify the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). After covalent binding of these modified primer analogs to the enzyme, the addition of [alpha-32P]dTTP, in the presence of a complementary template, led to elongation of the primer. This reaction was catalyzed by the active site of the enzyme carrying the covalently bound primer. The relative efficiency of labeling of the p66/p51 heterodimer compared to the p66/p66 and p51/p51 homodimers of HIV-1 RT was in agreement with the previously determined affinity of the various enzyme forms toward different primers. The analogues preferentially modified the p66 subunit of the HIV-1 RT heterodimer. The labeling of all RT forms by synthetic primer analogues showed significant and specific competition by the natural primer of HIV-1 RT, tRNA(Lys). In addition, the kinetics of inactivation of RT by primer analogues was studied. The affinity of the enzyme to those derivatives in the presence of poly(A) template was about 5-10 times higher than in the absence of template. Moreover, the maximal rates of HIV-1 RT inactivation by analogues in the absence of template were 3-4 times higher. Our results suggest that the mechanism of oligonucleotide primer binding to HIV-1 RT is different in the presence or absence of template.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1993

Male-sterility induction in transgenic tobacco plants with an unedited atp9 mitochondrial gene from wheat.

Cytoplasmic male sterility in plants is associated with mitochondrial dysfunction. We have proposed that a nuclear-encoded chimeric peptide formed by mitochondrial sequences when imported into the mitochondria may impair organelle function and induce male sterility in plants. A model developed to test this hypothesis is reported here. Assuming that the editing process in higher plant mitochondria reflects a requirement for producing active proteins, we have used edited and unedited coding sequences of wheat ATP synthase subunit 9 (atp9) fused to the coding sequence of a yeast coxIV transit peptide. Transgenic plants containing unedited atp9 exhibited either fertile, semifertile, or male-sterile phenotypes; controls containing edited atp9 or only the selectable marker gave fertile plants. Pollen fertility ranged from 31% to 75% in fertile plants, 10% to 20% in semifertile plants, and < 2% in male-sterile plants. Genetic and molecular data showed that the chimeric plasmid containing the transgene is inherited as a Mendelian trait. The transgenic protein is imported into the mitochondria. The production and frequency of semifertile or male-sterile transgenic plants conform to the proposed hypothesis.

Amino Acid Sequence↗
PubMed1993

Inhibition of the wheat germ DNA polymerase A activity by the antiviral drug HPA-23.

Wheat germ DNA polymerase A, a gamma-like enzyme, recognized efficiently natural and synthetic RNA templates, resembling a retroviral reverse transcriptase (P. Laquel et al., Biochim Biophys Acta 1048 (1990): 139-148). Ammonium-21-tungsto-9-antimoniate (HPA-23), an antiviral drug, inhibited the DNA polymerase A activities, independently of the template primers used, i.e. activated DNA or polyriboadenylic acid oligodeoxythymidylate (poly(rA)-oligo(dT)). The inhibition observed in the poly(rA)-oligo(dT)-directed DNA polymerase A activity occurred in the presence of either Mg2+ or Mn2+ as divalent cation, and also with the 2'-fluoro analogue of poly(rA) as template. HPA-23 was a non-competitive inhibitor with respect to TTP, activated DNA, poly(rA)-oligo(dT), and poly(dAfl)-oligo(dT). A preincubation study showed a reversible HPA-23 binding to DNA polymerase A, in the presence of poly(rA)-oligo(dT) as the template primer.

Antimony↗