Cloning and overexpression of Moloney murine leukemia virus reverse transcriptase in Escherichia coli

Kotewicz, Michael L.; D'Alessio, James M.; Driftmier, Katharine; Blodgett, Karen P.; Gerard, Gary F.

doi:10.1016/0378-1119(85)90003-4

Cited by 74 publications

(36 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This enzyme was predominantly insoluble when expressed in E. coli. Insertion of a termination codon earlier in the 3' sequence deleted a hydrophobic C-terminal region and resulted in the expression of a totally soluble protein (Kotewicz et al, 1985). This result implicates hydrophobic interactions in aggregation, consistent with results obtained when cloned viral glycoprotein genes are expressed in E. coli (Harris, 1984).…”

Section: Direct Expressionsupporting

confidence: 84%

The purification of eukaryotic polypeptides synthesized in Escherichia coli

Marston¹

1986

Biochemical Journal

858

379

View full text Add to dashboard Cite

Section: Direct Expressionsupporting

confidence: 84%

The purification of eukaryotic polypeptides synthesized in Escherichia coli

Marston¹

1986

Biochemical Journal

858

379

View full text Add to dashboard Cite

“…Unlike the OLA performed with PBMC DNA, the viral RNA from plasma must first be reverse transcribed to cDNA and then amplified by PCR. The reverse transcription step is not cycled, and thus the conversion of RNA to cDNA may be a fairly inefficient process (less than 50% with contaminating nucleic acids) (29). As discussed above, inefficient RNA-to-cDNA conversion may have been responsible for the reduced sensitivity of OLA with the plasma RNA sample.…”

Section: Resultsmentioning

confidence: 99%

Sensitive Oligonucleotide Ligation Assay for Low-Level Detection of Nevirapine Resistance Mutations in Human Immunodeficiency Virus Type 1 Quasispecies

et al. 2007

View full text Add to dashboard Cite

This study has adapted the oligonucleotide ligation assay (OLA) to probe for low-level nevirapine (NVP) resistance mutations K103N and Y181C in the human immunodeficiency virus type 1 (HIV-1) population of infected mother-infant pairs from Uganda. When NVP is used to prevent perinatal transmission, NVPresistant HIV-1 clones may be rapidly selected due to a low barrier for mutation and a relatively high level of fitness (compared to that of other drug-resistant HIV-1 clones). Monitoring for even a low frequency of NVP resistance mutations may help predict the success of subsequent treatment or warrant the use of another regimen to prevent transmission in a subsequent pregnancy. The standard OLA was optimized by using nonstandard bases in oligonucleotides to allow promiscuous base pairing and accommodate significant HIV-1 heterogeneity. Radiolabeled as opposed to fluorescently tagged oligonucleotides increased the sensitivity, whereas alteration of the template, oligonucleotides, salt, and thermostable DNA ligase concentrations increased the specificity for the detection of minority codons. This modified OLA is now capable of detecting mutants with the K103N or the Y181C mutation present in an HIV-1 population at a frequency of ϳ0.4% and is at least 10-to 30-fold more sensitive than the original protocol. A cohort of 19 Ugandan mothers who received NVP treatment perinatally were sampled 6 weeks postdelivery. Ten of 19 HIV-1 DNA samples extracted from peripheral blood mononuclear cells had a detectable K103N (0.5 to 44%) or Y181C (0.8 to 92.5%) mutation, but only one plasma HIV-1 RNA sample had a viral population with the Y181C mutation. These findings suggest that OLA is a robust, sensitive, and specific method for the detection of low-frequency drug resistance mutations in an intrapatient HIV-1 population.Three types of antiretroviral drugs (ARVs) are now used for the treatment of human immunodeficiency virus type 1 (HIV-1) infections, but only reverse transcriptase (RT) inhibitors are readily available to the vast majority of HIV-1-infected individuals in the developing world. The treatment regimen of choice is a combination of a nonnucleoside RT inhibitor (almost exclusively nevirapine [NVP]) and two nucleoside RT inhibitors, i.e., zidovudine (AZT) or stavudine plus lamivudine or didanosine.In addition to being the backbone of most treatment regimens, NVP is provided as a single dose to block the motherto-child transmission (MTCT) of HIV-1 in developing countries. In the absence of antiretroviral therapy, the frequency of MTCT is approximately 25 to 48% (2, 36, 38), whereas the administration of a short course of AZT therapy near the end of gestation (7,8,49) or the administration of a single dose of NVP at labor can reduce the rate of perinatal transmission to less than 20% (22,25,32,35,37). Although NVP remains very effective in the prevention of MTCT (22,32), the administration of even a single dose of NVP to

show abstract

“…First-strand complementary DNA was synthesized from 2 mg of total RNA with Moloney murine leukemia virus reverse transcriptase (Invitrogen) and oligo(dT) 12-18 primer (Invitrogen; Kotewicz et al, 1985). Gene-specific primers were designed to span two or more exons as listed in Supplemental Table S5.…”

Section: Rna Extraction and Quantitative Reverse Transcription-pcr Anmentioning

confidence: 99%

The Impact of the Branched-Chain Ketoacid Dehydrogenase Complex on Amino Acid Homeostasis in Arabidopsis

Peng¹,

Uygun²

2015

Plant Physiol.

View full text Add to dashboard Cite

The branched-chain amino acids (BCAAs) Leu, Ile, and Val are among nine essential amino acids that must be obtained from the diet of humans and other animals, and can be nutritionally limiting in plant foods. Despite genetic evidence of its importance in regulating seed amino acid levels, the full BCAA catabolic network is not completely understood in plants, and limited information is available regarding its regulation. In this study, transcript coexpression analyses revealed positive correlations among BCAA catabolism genes in stress, development, diurnal/circadian, and light data sets. A core subset of BCAA catabolism genes, including those encoding putative branched-chain ketoacid dehydrogenase subunits, is highly expressed during the night in plants on a diel cycle and in prolonged darkness. Mutants defective in these subunits accumulate higher levels of BCAAs in mature seeds, providing genetic evidence for their function in BCAA catabolism. In addition, prolonged dark treatment caused the mutants to undergo senescence early and overaccumulate leaf BCAAs. These results extend the previous evidence that BCAAs can be catabolized and serve as respiratory substrates at multiple steps. Moreover, comparison of amino acid profiles between mature seeds and darktreated leaves revealed differences in amino acid accumulation when BCAA catabolism is perturbed. Together, these results demonstrate the consequences of blocking BCAA catabolism during both normal growth conditions and under energy-limited conditions.The branched-chain amino acids (BCAAs) Leu, Ile, and Val are among nine amino acids essential for humans and other animals because they cannot be synthesized de novo (Harper et al., 1984). Plants synthesize BCAAs and are the main source of these essential nutrients in the diets of humans and agriculturally important animals. In addition to their nutritional value, BCAAs and BCAA-derived metabolites such as glucosinolates, fatty acids, and acyl sugars contribute to plant growth, development, defense, and flavor (Mikkelsen and Halkier, 2003;Taylor et al., 2004;Ishizaki et al., 2005;Slocombe et al., 2008;Araújo et al., 2010;Ding et al., 2012;Kochevenko et al., 2012).The BCAA biosynthetic pathway and its regulation have been investigated in Arabidopsis (Arabidopsis thaliana) and other plants for the past two decades, in large part because of the commercial importance of herbicides that inhibit acetohydroxy acid synthase, which is the committing enzyme of BCAA biosynthesis (Singh and Shaner, 1995;Aubert et al., 1997;Singh, 1999;McCourt et al., 2006;Tan et al., 2006;Binder, 2010;Chen et al., 2010;Yu et al., 2010). Strong correlations between the levels of free BCAAs were found in wildtype Arabidopsis seeds and tomato (Solanum lycopersicum) fruits Lu et al., 2008), which suggests coregulation of biosynthesis and/or degradation. This presumably is due, at least in part, to the fact that they share four common biosynthetic enzymes and three catabolic steps. However, despite long-term interest in the desirability of optim...

show abstract

Cloning and overexpression of Moloney murine leukemia virus reverse transcriptase in Escherichia coli

Cited by 74 publications

References 25 publications

The purification of eukaryotic polypeptides synthesized in Escherichia coli

The purification of eukaryotic polypeptides synthesized in Escherichia coli

Sensitive Oligonucleotide Ligation Assay for Low-Level Detection of Nevirapine Resistance Mutations in Human Immunodeficiency Virus Type 1 Quasispecies

The Impact of the Branched-Chain Ketoacid Dehydrogenase Complex on Amino Acid Homeostasis in Arabidopsis

Contact Info

Product

Resources

About