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The collection of gene expression data from human heart biopsies is important for understanding the cellular mechanisms of arrhythmias and diseases such as cardiac hypertrophy and heart failure. Many clinical and basic research laboratories conduct gene expression analysis using RNA from whole cardiac biopsies. This allows for the analysis of global changes in gene expression in areas of the heart, while eliminating the need for more complex and technically difficult single-cell isolation procedures (such as flow cytometry, laser capture microdissection, etc.) that require expensive equipment and specialized training. The abundance of fibroblasts and other cell types in whole biopsies, however, can complicate gene expression analysis and the interpretation of results. Therefore, we have designed a technique to quickly and easily purify cardiac myocytes from whole cardiac biopsies for RNA extraction. Human heart tissue samples were collected, and our purification method was compared with the standard nonpurification method. Cell imaging using acridine orange staining of the purified sample demonstrated that >98% of total RNA was contained within identifiable cardiac myocytes. Real-time RT-PCR was performed comparing nonpurified and purified samples for the expression of troponin T (myocyte marker), vimentin (fibroblast marker), and a-smooth muscle actin (smooth muscle marker). Troponin T expression was significantly increased, and vimentin and n-smooth muscle actin were significantly decreased in the purified sample (n = 8; P < 0.05). Extracted RNA was analyzed during each step of the purification, and no significant degradation occurred. These results demonstrate that this isolation method yields a more purified cardiac myocyte RNA sample suitable for downstream applications, such as real-time RTPCR, and allows for more accurate gene expression changes in cardiac myocytes from heart biopsies. real-time reverse transcriptase-polymerase chain reaction; vimentin; troponin T, smooth muscle actin; cardiomyocytes

The effects of copper (Cu) on trace metal and antibiotic resistance of Pseudomonas aeruginosa have been investigated. Cu treatments induced resistance not only to this metal but also, surprisingly, to zinc (Zn). Quantitative reverse transcription-PCR (qRT-PCR) revealed that after Cu treatment the transcription of the czcRS two-component system (TCS) operon was enhanced as well as that of the czcCBA operon encoding an efflux pump specific for zinc, cadmium, and cobalt. Cu treatments at the same time caused a decrease in the production of OprD porin, resulting in resistance to the carbapenem antibiotic imipenem. The CzcR regulator was known to repress oprD. However, Cu was still able to decrease the production of OprD and induce imipenem resistance in a czcRS knockout mutant. This strongly suggested that another Cu-dependent regulatory system was acting negatively on oprD expression. TCS regulator genes copR-copS have been shown to be involved in Cu tolerance in P. aeruginosa, qRT-PCR showed that overproduction of the CopR or of the CzcR regulator resulted in increased transcription of the czcC gene as well as in a decrease in oprD gene transcription, either in the wild-type strain or in the czcRS knockout mutant. Overproduction experiments suggest that a metal-dependent mechanism operates at the posttranscriptional level to control the production of the CzcCBA efflux pump. This study shows that CopR is a new negative regulator of OprD porin and that it links Zn, Cu, and imipenem resistances by interacting with the CzcRS TCS.

Bacillus subtilis has multiple stress response systems whose integrated action promotes growth and survival under unfavorable conditions. Here we address the function and transcriptional organization of a five-gene cluster containing ssrA, previously known to be important for growth at high temperature because of the role of its tmRNA product in rescuing stalled ribosomes. Reverse transcription-PCR experiments detected a single message for the secG-yvaK-rnr-smpB-ssrA cluster, suggesting that it constitutes an operon. However, rapid amplification of cDNA ends-PCR and lacZ fusion experiments indicated that operon transcription is complex, with at least five promoters controlling different segments of the cluster. One [[sigma].sup.A]-like promoter preceded secG ([P.sub.1]), and internal [[sigma].sup.A]-like promoters were found in both the rnr.smpB ([P.sub.2]) and smpB-ssrA intervals ([P.sub.3] and [P.sub.HS]). Another internal promoter lay in the secG-yvaK intercistronic region, and this activity ([P.sub.B]) was dependent on the general stress factor [[sigma].sup.B]. Null mutations in the four genes downstream from [P.sub.B] were tested for their effects on growth. Loss of yvaK (carboxylesterase E) or rnr (RNase R) caused no obvious phenotype. By contrast, smpB was required for growth at high temperature (52[degrees]C), as anticipated if its product (a small ribosomal binding protein) is essential for tmRNA (ssrA) function. Notably, smpB and ssrA were also required for growth at low temperature (16[degrees]C), a phenotype not previously associated with tmRNA activity. These results extend the known high-temperature role of ssrA and indicate that the ribosome rescue system is important at both extremes of the B. subtilis temperature range. doi:10.1128/JB.00062-.07

Staphylococcus epidermidis is both a human skin commensal and an opportunistic pathogen, causing infections linked to implanted medical devices. This paper describes localized tufts of fibrillar appendages on a subpopulation (25%) of wild-type (WT) S. epidermidis NCTC 11047 cells. The fibrils (122.2 [+ or -] 10.8 nm long) are usually in a lateral position on the cells. Fibriilar ([Fib.sup.+]) and nonfibrillar ([Fib.sup.-]) subpopulations were separated (enriched) by 34 sequential partitions of WT cells between a buffer phase and a hexadecane phase. Following enrichment, hydrophobic cells from the hexadecane phase comprised 70% [Fib.sup.-] cells and the less hydrophobic cells from the buffer phase entirely comprised [Fib.sup.-] cells. The [Fib.sup.+] and [Fib.sup.-] subpopulations did not revert on subculture (34 times) on solid medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell surface proteins from WT, [Fib.sup.+], and [Fib.sup.-] cells revealed two high-molecular-mass proteins (280 kDa and 230 kDa) on the WT and [Fib.sup.+] cells that were absent from the [Fib.sup.-] cells. Amino acid sequencing revealed that fragments of both the 280- and 230-kDa proteins had 100% identity to the accumulation-associated protein (Aap). Aap is known to cause biofilm formation if it is truncated by loss of the terminal A domain. Immunogoid staining with anti-Aap antibodies labeled tuft fibrils of the WT and [Fib.sup.+] cells but not the cell surface of Fibcells. The tufts were labeled with N-terminally directed antibodies (anti-A domain), showing that the fibrillar Aap was not truncated on the cell surface. Thus, the presence of full-length Aap correlated with the low biofilm-forming abilities of both WT and [Fib.sup.+] S. epidermidis NCTC 11047 populations. Reverse transcriptionPCR showed that aap was transcribed in both [Fib.sup.+] and [Fib.sup.-] cells. We therefore propose that full-length Aap is expressed on cells of S. epidermidis NCTC 11047 as tufts of short fibrils and that fibril expression is regulated at a posttranscriptional level.

Drug exporters contribute to the intrinsic drug resistance in many organisms. Although there are at least 20 exporter genes in Escherichia coli, most of them apparently do not confer drug resistance in complex laboratory media except for the AcrAB, EmrE, and MdfA efflux systems. In this study, we comprehensively investigated the growth phase-dependent expression of drug exporter genes. The expression of acrAB, emrAB, emrD, emrE, emrKY, mdfA, and ydgFE is stable at moderate levels during any growth phase, whereas mdtEF promoter activity greatly increased with cell growth and reached the maximum level at the late stationary phase. The growth phase-dependent increase in mdtEF expression was also observed on quantitative reverse transcription-PCR analysis. As expected from the transporter expression, the stationary-phase cells actually showed MdtEF-dependent tolerance to drugs and toxic dyes. Growth phase-dependent elevation of mdtEF expression was found to be mediated by the stationary-phase [sigma] factor rpoS and the RpoS-dependent signaling pathway, Hfq, GadY, and GadX. The induction level was decreased by tnaAB deletion, suggesting that indole sensing stimulates this process.

The potential relationship between helicase structural organization and unwinding mechanism is examined by performing in vitro Rho helicase experiments with model substrates containing an RNA-DNA helix downstream from a Rho loading site. The results have outlined the differences as well as the similarities in the mechanism of unwinding between Rho and other hexameric helicases, which are discussed in relation with the biological function of the Rho helicase.

The genetic mechanisms mediating the adaptation of Mycobacterium tuberculosis within the host are poorly understood. The best-characterized regulatory systems in this organism include sigma factors and two-component signal transduction systems, mprAB is a two-component system required by M. tuberculosis for growth in vivo during the persistent stage of infection. In this report, we demonstrate that MprAB is stress responsive and regulates the expression of numerous stress-responsive genes in M. tuberculosis. With DNA microarrays and quantitative real-time reverse transcription-PCR, genes regulated by MprA in M. tuberculosis that included two stress-responsive sigma factors were identified. Response regulator MprA bound to conserved motifs in the upstream regions of both sigB and sigE in vitro and regulated the in vivo expression of sigB and sigE in M. tuberculosis. In addition, mprA itself was induced following exposure to stress, establishing a direct role for this regulatory system in stress response pathways of M. tuberculosis. Induction of mprA and sigE by MprA in response to stress was mediated through the cognate sensor kinase MprB and required expression of the extracytoplasmic loop domain. These results provide the first evidence that recognition of and adaptation to specific stress in M. tuberculosis are mediated through activation of a two-component signal transduction system that directly regulates the expression of stress-responsive determinants.

The P2-1ike coliphages are highly similar; the structural genes show at least 96% identity. However, at two loci they have genes believed to be horizontally transferred. We show that the genetic content at the second loci, the TO region, contains six completely different sequences with high AT contents and with different open reading frames. The product of one of them exhibits reverse transcriptase activity and blocks infection of phage T5.

The Yersinia enterocolitica LuxI homologue YenI directs the synthesis of N-3-(oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL). In a Y. enterocolitica yenI mutant, swimming motility is temporally delayed while swarming motility is abolished. Since both swimming and swarming are flagellum dependent, we purified the flagellin protein from the parent and yenI mutant. Electrophoresis revealed that in contrast to the parent strain, the yenI mutant grown for 17 h at 26[degrees]C lacked the 45-kDa flageilin protein FleB. Reverse transcription-PCR indicated that while mutation of yenI had no effect on yenR, flhDC (the motility master regulator) orfliA (the flagellar sigma factor) expression, fleB (the flagellin structural gene) was down-regulated. Since 3-oxo-C6-HSL and C6-HSL did not restore swimming or swarming in the yenI mutant, we reexamined the N-acylhomoserine lactone (AHL) profile of Y. enterocolitica. Using AHL biosensors and mass spectrometry, we identified three additional AHLs synthesized via YenI: N-(3-oxodecanoyl)homoserine lactone, N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL), and N-(3-oxotetradecanoyl)homoserine lactone. However, none of the long-chain AHLs either alone or in combination with the short-chain AHLs restored swarming or swimming in the yenI mutant. By investigating the transport of radiolabeled 3-oxo-C12-HSL and by introducing an AHL biosensor into the yenI mutant we demonstrate that the inability of exogenous AHLs to restore motility to the yenI mutant is not related to a lack of AHL uptake. However, both AHL synthesis and motility were restored by complementation of the yenI mutant with a plasmid-borne copy of yenI.

In the murine model of infection, a Mycobacterium tuberculosis mce1 operon mutant elicits an aberrant granulomatous response, resulting in uncontrolled replication and failure to enter a persistent state. In this study, we demonstrate that the mce1 genes can be transcribed as a 13-gene polycistronic message encompassing Rv0166 to Rv0178. Quantitative reverse transcriptase PCR and immunoblot analyses revealed that the mce1 genes and proteins are expressed during in vitro growth but are significantly down-regulated in intracellular bacilli isolated from murine macrophages. A homologue of the FadR subfamily of GntR transcriptional regulators, Rv0165c (designated Mce1R), lies upstream and is divergently transcribed from the operon. To investigate whether this gene plays a role in regulation of mce1 expression, we created an M. tuberculosis mce1R deletion mutant. There was no difference in expression of mce1 operon genes in [DELTA]mce1R compared to expression in the wild type during logarithmic growth in vitro. However, in bacilli isolated from murine macrophages, expression of mce1 genes was significantly higher in [DELTA]mce1R. In addition, overexpression of mce1R resulted in repression of the mce1 genes. These data demonstrate that Mce1R is a negative regulator that acts intracellularly to repress expression of the mce1 operon. We propose that Mce1R facilitates balanced temporal expression of the mce1 products required for organized granuloma formation, which is both protective to the host and necessary for the persistence of M. tuberculosis.

The distribution of the metalloprotease gene zmpA was determined among strains of the Burkholderia cepacia complex (Bcc). The zmpA gene was present in B. cepacia, B. cenocepacia, B. stabilis, B. ambifaria and B. pyrrocinia but absent from B. multivorans, B. vietnamiensis, B. dolosa, and B. anthina. The presence of zmpA generally correlated with extracellular proteolytic activity with the exception of five strains, which had zmpA but had no detectable proteolytic activity when skim milk agar was used as a substrate (zmpA protease deficient). Western immunoblot experiments with anti-ZmpA antibodies suggest that the zmpA protease-deficient strains do not secrete or accumulate detectable ZmpA. Transcriptional zmpA::lacZ fusions were introduced in selected strains of the Bcc. zmpA::lacZ was expressed in all strains, but expression was generally lower in the zmpA protease-deficient strains than in the zmpA protease-proficient strains. Quantitative reverse transcriptase real-time PCR demonstrated that zmpA protease-deficient strains did express zmpA mRNA, although at various levels. ZmpA has previously been shown to be positively regulated by the CepIR quorum-sensing system. Addition of exogenous AHLs did not restore extracellular protease production to any of the zmpA protease-deficient strains; however, introduction of cepR in trans complemented protease activity in two of five strains. Extra-cellular proteolytic activity was restored by the presence of zmpA in trans in two of the five strains. These studies suggest that although some strains of the Bcc contain the zmpA gene, multiple factors may influence its expression.

The steady-state kinetic measurements are extended to two replicative DNA polymerases, exonuclease-deficient T7 DNA polymerase T7(super -) and HIV reverse transcriptive (HIV-RT). The influences of G[8-5]C on the thermal stability and base pairing properties of DNA duplexes are examined.

Bacterial gene expression was studied during in vitro exponential and early stationary growth in medium with and without sufficient iron, respectively. The aims of the investigation were to develop and test a sensitive and reproducible method for the study of gene expression in Actinobacillus pleuropneumoniae by real-time quantitative reverse transcription (RT)-PCR and to evaluate a number of suitable internal controls.

The study developed broadly reactive one-step TaqMan reverse transcription (RT)-PCR assays for the detection of genogroup I (GI) and GII noroviruses (NoV) in fecal samples, as well as shellfish samples. These assays provide rapid, sensitive, and reliable detection of NoV and should prove to be useful for routine monitoring of both clinical ad shellfish samples.

An approach for inhibiting the ribonuclease H (RNase H) activity of human immunodeficiency virus type I (HIV-I) reverse transcriptase (RT) is demonstrated by targeting RNase H RNA.DNA hybrid substrates. In this approach the binding of the 4,5-disubstituted 2-deoxystreptamine aminoglycosides, neomycin, paromomycin and ribostamycin, to two different chimeric RNA-DNA duplexes inhibits specific RT-mediated RNase cleavage in competitive manner.

Research has been conducted on the YXDD motif of the reverse transcriptase family. The evidence that reverse transcriptases catalyze DNA synthesis is presented.

The HIV-1 reverse transcriptase-RNA/RNA-dCTP ternary complex has a different conformation than complexes involving DNA/DNA and DNA/RNA. Because of this, dCTP incorporation is significantly less than that for DNA/DNA and DNA/RNA substrates.

Researchers developed a synthetic peptide that inhibits the conformational change that HIV reverse transcriptase undergoes during dimerization. The enzyme is a heterodimer and the thumb domain of p51 is essential for dimerization.

Amino terminal domains in the yeast telomerase catalytic subunit are discussed with consideration of their essential functions shown by selection for viable mutants. A large library having in it random mutations in the amino terminus of the EST2 gene, the one that encodes the Saccharomyces cerevisiae telomerase reverse transcriptases (TERTs), was set up. Certain functional alleles were selected by ability to rescue senescence of telomerase-negative cells. Analysis was carried out on 265 mutations.

Research was conducted to identify the inhibitors of DNA strand transfer reactions. A chemical mass screening assay was created using a novel application of scintillation proximity technology that was specifically aimed at detecting inhibition of one or more steps of the DNA strand transfer process. The identification of a broad range of inhibitors against DNA strand transfer indicates that the complex reaction could be an important target for drug discovery and development.

Telomere shortening and apoptosis are discussed as seen in telomerase-inhibited human tumor cells. The role of telomerase activity in proliferation of human tumor and immortal cells has been looked at using inhibition of telomerase reverse transcriptase (TERT) function. Stable cell lines were generated in two telomerase-positive human cell lines to look at effects of long-term expression of the dominant-negative mutants on tumor cells.

Research was conducted to illustrate the synthesis of multiple extracellular lipases in Candida rugosa. To quantify the expression of lip genes, a competitive reverse transcription-PCR assay was developed. Using the approach made it possible to demonstrate the differential expression of the five lip genes in the presence of different inducers that are known to be able to increase C rugosa lipase production.

A reverse transcription-PCR assay was developed to investigate the expression of Tri5 by Fusarium species in planta and in vitro. An increase in Tri5 expression was observed following treatment of Fusarium culmorum with fungicides. An inverse relationship between Tri5 expression and biomass was also observed. Results provide an enhanced understanding of the influence of different host tissues and chemical control agents on trichothecene production.

Most of the pre-steady-state kinetic studies of HIV-1 reverse transcriptase (HIV-1 RT) have shown substoichiometric bursts of product formation in the initial reaction cycle. The extinction coefficient of the HIV-1 RT heterodimer was altered by applying quantitative amino acid analysis. It was observed that normal nucleotide incorporation occurs with quantitative bursts in the first cycle. Findings showed that HIV-1 RT is fully active in normal nucleotide incorporation and that substoichiometric bursts with altered systems are well-defined by the presence of nonproductive ternary complexes, which can isomerize to productive complexes.

Research was conducted to examine the kinetics for the incorporation of dCMP, ddCMP and (+)SddCMP and its unnatural beta-L-(-) enantiomer, (-)SdCMP, into DNA/DNA and DNA/RNA primer-templates catalyzed by HIV-1 RT. The objective was to gain an insight into the differences between RNA-dependent and DNA-dependent polymerization. Results suggest that perturbations on the ribose ring of cytidine analogues decrease the rate and efficiency of incorporation but improves the binding affinity.

The multifunctional HIV reverse transcriptase (RT) enzyme catalyzes RNA-dependent DNA polymerase, DNA-dependent DNA polymerase and RNase H activities. Four crystallographic analyses of HIV-1 RT are reported in complexes with different carboxailides, with the aim of gaining an understanding of the differing inhibition of mutant RTs, and clarifying previous studies.

The topography and functional features of the complex produced in vitro between human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and its primer tRNA(super Lys3) were investigated. Chimeric primers having tRNA(super Lys3) linked to U and T residues of various lengths were synthesized. Results showed that the affinity of the oligonucleotide primers for HIV-1 RT is drastically enhanced when linked to primer tRNA.

The reason for the seemingly slow, nonprocessive polymerization reaction catalyzed by HIV-1 reverse transcriptase (RT) during transcription initiation, when both the primer and the template are made of RNA, was investigated through single-turnover and equilibrium measurements. Results showed that binding and not catalytic characteristics of the initiation p/ts cause the overall slow, nonprocessive polymerization rate during initiation.

The kinetics of 8-oxo-7,8-dihydroguanosine triphosphate (8-oxo-dGTP) incorporation into DNA by Escherichia coli polymerases I exo- (KF-) and II exo- (Pol II-), HIV-1 reverse transcriptase (HIV-1 RT) and bacteriophage T7 exo- (T7-) were investigated. The goal was to examine the misincorporation potential for 8-oxo-dGTP and to study the function of base pairing symmetry in DNA polymerase fidelity. Results indicated that polymerase fidelity does not rely only on the overall geometry of Watson-Crick base pairs and the enzyme binding site is asymmetrical.

A new study compares the differing modes of DNA synthesis used by heterodimeric equine infectious anemia virus (EIAV) p66/51 and homodimeric EIAV p51/51. Analysing the dimerization behaviour shows that the dimer stability of HIV-1 reverse transcriptase enzyme is lower than that of EIAV transcriptase. It is suggested that the homodimetric enzyme forms a conformation similar to p66/51.

RNA primers initiate plus-strand synthesis and minus-strand synthesis of HIV replication. A new study analyses the structure of the 3'-DNA-RNA-5' junction formed during the commencement of minus-strand synthesis of HIV replication. It is shown that the hybrid octamer which corresponds to the DNA-RNA junction adopts a global A-type conformation throughout its length.

A study was conducted to examine the possible function of the p51 subunit of HIV-1 reverse transcriptase (RT) in the template-primer binding process. The effects of acetonitrile addition on the polymerase activity of HIV-1 RT were also studied before and after the templaten primer binding step. Results indicated that acetonitrile dissociated the subunits of the HIV-1 RT into catalytically inactive monomers.

A study was conducted to analyze the fidelity of mutant HIV-1 reverse transcriptases using random sequence mutagenesis and complementation in a bacterial system. A complementation system in Escherichia coli that allowed genetic selection for DNA-dependent DNA polymerase activity of HIV RT was used in the study. Results suggested mechanisms controlling the replication accuracy of HIV RT based on drug-resistant mutants.

Two protein components of telomerase, p80 and p95, from Tetrahymena thermophila, were identified by their involvement with telomerase activity and telomerase RNA. Recombinant versions of the proteins were produced to study functions in the ribonucleoprotein. They can form a complex that is independent of RNA. The DNA binding properties of p95 show it interacts with telomeric substrate DNAs with specificity characteristic of the Tetrahymena telomerase substrate anchor site.

The reverse transcriptase/polymerase chain reaction (RT-PCR) process is examined with particular attention to the common observation that control DNA reaction mixtures with RT do not provide DNA amplification during PCR while control DNA reaction mixtures without RT do not inhibit PCR. The findings indicate that the RT interaction with the specific mRNA or cDNA template mediates RT inhibition of the PCR and that such inhibitory effect is influenced by template concentration.

The effect of RNA secondary structure switching in in vitro replication of the human immunodeficiency virus reverse transcriptase was investigated through the use of rapid kinetic methods. Results obtained showed that the RNA secondary structure switching mediated the translational regulation, protein synthesis and mRNA splicing. Also, the switching played an important role in replication catalyzed by the HIV-RT in vitro.

It is possible that the RNA dependent DNA replication event plays a role in the generation of 3'-azido-3'-deoxythymidine (AZT) resistance. Research indicates that RNA dependent DNA polymerization function of reverse transcriptase (RT) may play a more significant role in the generation of resistance to AZTTP than DNA dependent DNA polymerization. No differences were seen in the cleavage patterns of the degraded RNA template for mutant RT and wild type. This finding does not support the possibility of an altered binding mode for the mutant compared to wild type RT.

Research designed to compare replication fidelity of HIV-1 reverse transcriptase (RT1) using DNA/DNA and RNA/DNA template-primer substrates has established that there are differences between DNA and RNA substrates. RT showed high fidelity, indicating that the high degree of genomic variation of HIV-1 may be attributable more to a defense mechanism of the virus to escape the surveillance of the host immune system than to insufficient regulation on the part of RT.

The thiocarboxanilide UC781 was studied to determine its use in the retrovirucidal/microbiocidal formulations in preventing HIV sexual transmission from infected individuals to noninfected individuals. Results reveal that thiocarboxanilde UC781 is a tight-binding inhibitor of HIV-1 RT. The treatment of UC781 with isolated HIV-1 virions leads to virus ineffectivity. This suggests that the compound is useful in the formulation of retrovirucidal.

The nucleotide conformational alteration in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) was studied using nondenaturing gel electrophoresis. Dead-end complex was formed in the presence of deoxynucleotide triphosphate (dNTP) between dideoxynucleotide chain-terminated primer, HIV-1 RT and DNA template. Dead-end complex formation by a complementary nucleotide was a minimum of 2000-times more efficient than with ribonucleotides or noncomplementary dNTP's.

The contribution of primer grip residues of human immunodeficiency virus type 1 reverse transcriptase to its palm subdomain architecture and nearby structural elements was studied by analyzing the ribonuclease H and DNA polymerase activities of enzymes where aromatic substitutions had been made at Tyr232 and Trp229. Research suggests that oligoribo- and oligodeoxyribonucleic acid primers may be discriminated between as a result of small changes to primer grip architecture.

Enzymatic profiles of recombinant human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) with five mutations (A62V, V75I, F77L, F116Y and Q151M) which confers resistance to multiple 2',3'-dideoxynucleosides (ddNs) on HIV-1 were examined. Findings indicated that RT(sub A62V/V75I/F77L/F116Y/Q151M) is insensitive to ddATP, AZTTP, D4TTP, FddATP and ddCTP but is insensitive to PMEApp, 3TCTP and FddCTP.

Reverse transcriptases (RTs) have a significant role in the propagation of several genetic elements. They duplicate the RNA genomes of retroviruses and long terminal repeat transposons into double stranded DNA that has to be integrated into the host genome to ensure proliferation. Long Interspersed Nucleotide Element-like materials are non-long terminal repeat retrotransposons that also require RT.

Site directed mutagenesis of Arg 110 was performed, replacing it with Ala, Lys and Glu to determine whether Arg 110 of murine leukemia virus (MuLV) reverse transcriptase (RT) is a significant contributor to the catalytic mechanism of MuLV RT. Findings indicated that the rate limiting step for the mutant enzymes may be before and after the phosphodiester bond formation and that Arg 110 of MuLV RT contributes to the conformational change steps before and after the chemical step of polymerase reaction.

Five classic DNA minor groove-binding drugs and a series of bis-linked lexitropsins based on netropsin and distamycin were screened for their effectiveness in inhibiting transcription by HIV-1 reverse transcriptase on a poly(rA)*oligo template-primer (TP). The two most effective drugs, 3,5 m-pyridyl-linked bisdistamycin and trans-vinyl-linked bisdistamycin, indicated improved inhibition in reactions started with pre-incubated enzyme template-primer and reduced affinity for a free TP analog, when compared with the parent drug distamycin.

Amplification of oligonucleotide repeats may be due to an aberration in the copying process of polymerases. It is shown that in the presence of MnCl2, mismatch formation commits HIV-1 reverse transcriptase to a new mode of DNA synthesis which yields repetitive products. This activity is distinct from terminal transferase since it needs specific DNA motifs in the template. This mechanism also works on homologous RNA templates where it produces reiterative products more than 150 nucleotides long.

Alterations in Leu(super 234) in the primer grip of the p66 subunit of the human immunodeficiency virus 1 reverse transcriptase produces a subunit that fails to associate into a heterodimer. Mutations in any residue between Trp(super 229) and Tyr(super 232) change the functions associated with DNA polymerase and ribonuclease H domains. An analysis of retroviral sequences indicates that aromatic residues are preferred at positions 229 and 232. The residues probably interact and position the template-primer, and supply the architecture necessary for primer grip.