Moloney Murine Leukemia Virus Reverse Transcriptase Research Articles

Structural and Inhibition Studies of the RNase H Function of Xenotropic Murine Leukemia Virus-Related Virus Reverse Transcriptase

RNase H inhibitors (RNHIs) have gained attention as potential HIV-1 therapeutics. Although several RNHIs have been studied in the context of HIV-1 reverse transcriptase (RT) RNase H, there is no information on inhibitors that might affect the RNase H activity of other RTs. We performed biochemical, virological, crystallographic, and molecular modeling studies to compare the RNase H function and inhibition profiles of the gammaretroviral xenotropic murine leukemia virus-related virus (XMRV) and Moloney murine leukemia virus (MoMLV) RTs to those of HIV-1 RT. The RNase H activity of XMRV RT is significantly lower than that of HIV-1 RT and comparable to that of MoMLV RT. XMRV and MoMLV, but not HIV-1 RT, had optimal RNase H activities in the presence of Mn²⁺ and not Mg²⁺. Using hydroxyl-radical footprinting assays, we demonstrated that the distance between the polymerase and RNase H domains in the MoMLV and XMRV RTs is longer than that in the HIV-1 RT by ∼3.4 Å. We identified one naphthyridinone and one hydroxyisoquinolinedione as potent inhibitors of HIV-1 and XMRV RT RNases H with 50% inhibitory concentrations ranging from ∼0.8 to 0.02 μM. Two acylhydrazones effective against HIV-1 RT RNase H were less potent against the XMRV enzyme. We also solved the crystal structure of an XMRV RNase H fragment at high resolution (1.5 Å) and determined the molecular details of the XMRV RNase H active site, thus providing a framework that would be useful for the design of antivirals that target RNase H.

Neurotensin Triggers Dopamine D2 Receptor Desensitization through a Protein Kinase C and β-Arrestin1-dependent Mechanism

The peptide neurotensin (NT) is known to exert a potent excitatory effect on the dopaminergic system by inhibiting D2 dopamine (DA) receptor (D2R) function. This regulation is dependent on activation of PKC, a well known effector of the type 1 NT receptor (NTR1). Because PKC phosphorylation of the D2R has recently been shown to induce its internalization, we hypothesized that NT acts to reduce D2R function through heterologous desensitization of the D2R. In the present study, we first used HEK-293 cells to demonstrate that NT induces PKC-dependent D2R internalization. Furthermore, internalization displayed faster kinetics in cells expressing the D2R short isoform, known to act as an autoreceptor in DA neurons, than in cells expressing the long isoform, known to act as a postsynaptic D2R. In patch clamp experiments on cultured DA neurons, overexpression of a mutant D2S lacking three key PKC phosphorylation sites abrogated the ability of NT to reduce D2R-mediated cell firing inhibition. Short interfering RNA-mediated inhibition of β-arrestin1 and dynamin2, proteins important for receptor desensitization, reduced agonist-induced desensitization of D2R function, but only the inhibition of β-arrestin1 reduced the effect of NT on D2R function. Taken together, our data suggest that NT acutely regulates D2 autoreceptor function and DA neuron excitability through PKC-mediated phosphorylation of the D2R, leading to heterologous receptor desensitization.

Mechanistic insights into the roles of three linked single-stranded template binding residues of MMLV reverse transcriptase in misincorporation and mispair extension fidelity of DNA synthesis

To obtain some insights into the structure–function relationship of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT), we modeled the catalytic state ternary complexes of this protein using the corresponding RT from human immunodeficiency virus type 1 (HIV-1) and available structures of MMLV RT. We observed that three MMLV RT single-stranded template binding residues, Y64, D114, and R116, act as a linked set through mutual interactions, including hydrogen bonding and ion-pairing. The analogous residues of HIV-1 RT have a somewhat different environment and they lack this linked phenomenon. To understand the functional implication of this linked set of MMLV RT, we performed site-directed mutagenesis at these three positions. Then the mutant enzymes were examined for their biochemical properties and nucleotide selectivity. Mutagenesis of these residues (Y64A, D114A, and R116A) resulted in enzymes with slight to modest changes in polymerase activities. The processivity of DNA synthesis correlated positively with the binding affinity of the MMLV RT variants. Lower fidelity in mutants was indicated by measurements of misincorporation and mispair extension fidelity of wild type (WT) and mutant RTs, in contrast to earlier works that indicate that mutations at the analogous positions in HIV-1 RT result in relatively higher fidelity. These data together with structural analysis suggest that this structural set may therefore be a key factor responsible for the different fidelity of these two RTs.

Effects of Organic Solvents on the Reverse Transcription Reaction Catalyzed by Reverse Transcriptases from Avian Myeloblastosis Virus and Moloney Murine Leukemia Virus

The use of certain organic chemicals has been found to improve yields and specificity in PCR. In this study, we examined the effects of dimethyl sulfoxide (DMSO), formamide, and glycerol on the reverse transcription reaction catalyzed by reverse transcriptases (RTs) from avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (MMLV). At 42 °C, DMSO at 24% v/v and formamide at 12-14% inhibited the cDNA synthesis reaction, but DMSO at 12% and formamide at 6-8% improved the efficiency of the cDNA synthesis reaction at low temperatures (25-34 °C). Glycerol at 10% improved the efficiency of the cDNA synthesis reaction at high temperatures (49-61 °C). The effects of DMSO and formamide appeared to be accompanied by decreases in the melting temperatures of the primers, and the effect of glycerol was due to increases in the thermal stabilities of AMV RT and MMLV RT.

Temporal expression pattern of insulin-like growth factors (IGF-1 and IGF-2) ligands and their receptors (IGF-1R and IGF-2R) in buffalo ( Bubalus bubalis) embryos produced in vitro

The objective of the study was to assess the temporal expression of genes for IGF-1, IGF-2 and their receptors in different pre-implantation stage buffalo embryos (from oocytes to blastocyst) and to evaluate the effect of IGF-1 on culture media during in vitro buffalo embryo development. Buffalo oocytes were retrieved from slaughterhouse derived ovaries. In vitro matured oocytes were fertilized with frozen buffalo bull semen. Presumptive zygotes were cultured in modified synthetic oviductal fluid (mSOF supplemented with β-mercaptoethanol (100 μM) and without (control) or with IGF-1 (100 ng/ml) till blastocyst stage. For each treatment five replicates were taken. Supplementation of IGF-1 to embryo culture media resulted in significantly higher blastocyst formation rate (29.7% versus 25.6%; p < 0.05) than the control group. In vitro produced embryos of different stages were stored in small amount of PBS at − 70 °C till used for RNA isolation. Total RNA isolated from oocytes and pre-implantation embryos was reverse transcribed to cDNA in total volume of 25 μl reaction mixture using Moloney-Murine Leukemia Virus Reverse Transcriptase (MMLV-RT). Polymerase chain reaction (PCR) was performed using cDNA from different pools of oocytes and embryos. RT-PCR revealed that IGF-1 mRNA was neither expressed in oocytes (immature, matured and matured vitrified oocytes) nor at any pre-implantation stage embryos while mRNA for IGF-1R, IGF-2 and IGF-2R were detected in all oocytes as well as in all pre-implantation stage embryos. It can be concluded that supplementation of IGF-1 is useful for in vitro oocyte maturation and embryo development in buffalo.

Molecular Identification of Carnosine Synthase as ATP-grasp Domain-containing Protein 1 (ATPGD1)

Carnosine (beta-alanyl-L-histidine) and homocarnosine (gamma-aminobutyryl-L-histidine) are abundant dipeptides in skeletal muscle and brain of most vertebrates and some invertebrates. The formation of both compounds is catalyzed by carnosine synthase, which is thought to convert ATP to AMP and inorganic pyrophosphate, and whose molecular identity is unknown. In the present work, we have purified carnosine synthase from chicken pectoral muscle about 1500-fold until only two major polypeptides of 100 and 90 kDa were present in the preparation. Mass spectrometry analysis of these polypeptides did not yield any meaningful candidate. Carnosine formation catalyzed by the purified enzyme was accompanied by a stoichiometric formation, not of AMP, but of ADP, suggesting that carnosine synthase belongs to the "ATP-grasp family" of ligases. A data base mining approach identified ATPGD1 as a likely candidate. As this protein was absent from chicken protein data bases, we reconstituted its sequence from a PCR-amplified cDNA and found it to fit with the 100-kDa polypeptide of the chicken carnosine synthase preparation. Mouse and human ATPGD1 were expressed in HEK293T cells, purified to homogeneity, and shown to catalyze the formation of carnosine, as confirmed by mass spectrometry, and of homocarnosine. Specificity studies carried out on all three enzymes were in agreement with published data. In particular, they acted with 15-25-fold higher catalytic efficiencies on beta-alanine than on gamma-aminobutyrate. The identification of the gene encoding carnosine synthase will help for a better understanding of the biological functions of carnosine and related dipeptides, which still remain largely unknown.

Multiple Nucleotide Preferences Determine Cleavage-Site Recognition by the HIV-1 and M-MuLV RNases H

The RNase H activity of reverse transcriptase is required during retroviral replication and represents a potential target in antiviral drug therapies. Sequence features flanking a cleavage site influence the three types of retroviral RNase H activity: internal, DNA 3′-end-directed, and RNA 5′-end-directed. Using the reverse transcriptases of HIV-1 (human immunodeficiency virus type 1) and Moloney murine leukemia virus (M-MuLV), we evaluated how individual base preferences at a cleavage site direct retroviral RNase H specificity. Strong test cleavage sites (designated as between nucleotide positions − 1 and + 1) for the HIV-1 and M-MuLV enzymes were introduced into model hybrid substrates designed to assay internal or DNA 3′-end-directed cleavage, and base substitutions were tested at specific nucleotide positions. For internal cleavage, positions + 1, − 2, − 4, − 5, − 10, and − 14 for HIV-1 and positions + 1, − 2, − 6, and − 7 for M-MuLV significantly affected RNase H cleavage efficiency, while positions − 7 and − 12 for HIV-1 and positions − 4, − 9, and − 11 for M-MuLV had more modest effects. DNA 3′-end-directed cleavage was influenced substantially by positions + 1, − 2, − 4, and − 5 for HIV-1 and positions + 1, − 2, − 6, and − 7 for M-MuLV. Cleavage-site distance from the recessed end did not affect sequence preferences for M-MuLV reverse transcriptase. Based on the identified sequence preferences, a cleavage site recognized by both HIV-1 and M-MuLV enzymes was introduced into a sequence that was otherwise resistant to RNase H. The isolated RNase H domain of M-MuLV reverse transcriptase retained sequence preferences at positions + 1 and − 2 despite prolific cleavage in the absence of the polymerase domain. The sequence preferences of retroviral RNase H likely reflect structural features in the substrate that favor cleavage and represent a novel specificity determinant to consider in drug design.

Increased Thermostability and Fidelity of DNA Synthesis of Wild-Type and Mutant HIV-1 Group O Reverse Transcriptases

Reverse transcription coupled with DNA amplification has become a well-established and powerful molecular technique for studying ribonucleic acids. However, the efficiency of those reactions is largely dependent on the molecular properties of currently used reverse transcriptases (RTs). Engineered and natural RT variants with improved thermostability and fidelity of DNA synthesis should be of great utility in the amplification of RNA targets. In this study, we demonstrate that the wild-type (WT) HIV-1 group O (O_WT) RT shows increased thermostability in comparison with Moloney murine leukemia virus RT and a prototypic HIV-1 group M:subtype B (BH10_WT) RT, while rendering higher yields in reverse transcription PCRs that included a cDNA synthesis step performed at a high temperature range (57–69 °C). In addition, the O_WT RT showed 2.5-fold increased accuracy in M13 lacZα forward mutation assays in comparison with the BH10_WT RT. Unlike the BH10_WT enzyme, O_WT RT showed a very low error rate for frameshifts. Mutational hot spots induced by O_WT RT occurred at nucleotide runs, suggesting a dislocation-mediated mechanism for the generation of base substitutions. In HIV-1 group O RT, substituting Ile75 for Val rendered an enzyme that was 1.9 and 4.7 times more faithful than O_WT RT and BH10_WT RTs, respectively, in forward mutation assays. The mutant RT also showed increased misinsertion and mispair extension fidelity in kinetic assays. However, its mutational spectrum was similar to that obtained with the WT group O polymerase. V75I caused a loss of efficiency of reverse transcription PCR amplifications at 65 and 68 °C in comparison with O_WT RT. However, a double mutant devoid of RNase H activity (V75I/E478Q) was found to reverse-transcribe at temperatures as high as 68 °C, while maintaining the increased accuracy of the V75I mutant.

A method for quantifying melanosome transfer efficacy from melanocytes to keratinocytes in vitro

Several different in vivo and in vitro bioassays are used to evaluate melanosome transfer efficacy from melanocytes to keratinocytes. However, these methods are complicated and time consuming. Here, we report on a simple, rapid, direct, and reliable in vitro method for observing the process of melanosome transfer from melanocytes to keratinocytes. First, we selected and tested a melanoma cell line RPMI-7951 that can normally synthesize melanin and transfer from mature melanosomes to keratinocytes in vitro. We cocultured these cells with a human ovarian teratoma transformed epidermal carcinoma cell line, which is also capable of accepting melanosomes transferred from melanocytes, as in normal keratinocytes. The cells were cocultured for 24-72 h and double labeled with FITC-conjugated antibody against the melanosome-associated protein TRP-1, and with Cy5-conjugated antibody against the keratinocyte-specific marker keratin 14. The cells were examined by fluorescence microscope and flow cytometry. Melanosome transfer from melanocytes to keratinocytes increased in a time-dependent manner. To verify the accessibility of this method, the melanosome transfer inhibitor, a serine protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, and a melanosome transfer stimulator, alpha-melanocyte-stimulating hormone, were added. The serine protease inhibitor decreased melanosome transfer, and alpha-melanocyte-stimulating hormone increased melanosome transfer, in a dose-dependent manner. In conclusion, this is a simple, rapid, and effective model system to quantify the melanosome transfer efficacy from melanocytes to keratinocytes in vitro.

Iodine Deficiency Induces a Thyroid Stimulating Hormone-Independent Early Phase of Microvascular Reshaping in the Thyroid

Expansion of the thyroid microvasculature is the earliest event during goiter formation, always occurring before thyrocyte proliferation; however, the precise mechanisms governing this physiological angiogenesis are not well understood. Using reverse transcriptase-polymerase chain reaction and immunohistochemistry to measure gene expression and laser Doppler to measure blood flow in an animal model of goitrogenesis, we show that thyroid angiogenesis occurred into two successive phases. The first phase lasted a week and involved vascular activation; this process was thyroid-stimulating hormone (TSH)-independent and was directly triggered by expression of vascular endothelial growth factor (VEGF) by thyrocytes as soon as the intracellular iodine content decreased. This early reaction was followed by an increase in thyroid blood flow and endothelial cell proliferation, both of which were mediated by VEGF and inhibited by VEGF-blocking antibodies. The second, angiogenic, phase was TSH-dependent and was activated as TSH levels increased. This phase involved substantial up-regulation of the major proangiogenic factors VEGF-A, fibroblast growth factor-2, angiopoietin 1, and NG2 as well as their receptors Flk-1/VEGFR2, Flt-1/VEGFR1, and Tie-2. In conclusion, goiter-associated angiogenesis promotes thyroid adaptation to iodine deficiency. Specifically, as soon as the iodine supply is limited, thyrocytes produce proangiogenic signals that elicit early TSH-independent microvascular activation; if iodine deficiency persists, TSH plasma levels increase, triggering the second angiogenic phase that supports thyrocyte proliferation.

Sequential Regulation of Diacylglycerol Acyltransferase 2 Expression by CAAT/Enhancer-binding Protein β (C/EBPβ) and C/EBPα during Adipogenesis

Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step of triacylglycerol (TG) synthesis. Despite the existence of an alternative acyltransferase (DGAT1), mice lacking DGAT2 have a severe deficiency of TG in adipose tissue, indicating a nonredundant role for this enzyme in adipocyte TG synthesis. We have studied the regulation of DGAT2 expression during adipogenesis. In both isolated murine preadipocytes and 3T3-L1 cells the temporal pattern of DGAT2 expression closely mimicked that of genes whose expression is regulated by CAAT/enhancer-binding protein beta (C/EBPbeta). Inhibition of C/EBPbeta expression in differentiating preadipocytes reduced DGAT2 expression, and electrophoretic mobility shift assay and chromatin immunoprecipitation experiments identified a promoter element in the DGAT2 gene that is likely to mediate this effect. The importance of C/EBPbeta in adipocyte expression of DGAT2 was confirmed by the finding of reduced DGAT2 expression in the adipose tissue of C/EBPbeta-null animals. However, DGAT2 expression is maintained at high levels during the later stages of adipogenesis, when C/EBPbeta levels decline. We show that, at these later stages of differentiation, C/EBPalpha is capable of substituting for C/EBPbeta at the same promoter element. These observations provide novel insight into the transcriptional regulation of DGAT2 expression. Moreover, they further refine the complex and serial roles of the C/EBP family of transcription factors in inducing and maintaining the metabolic properties of mature adipocytes.

Escherichia coli DNA polymerase III ε subunit increases Moloney murine leukemia virus reverse transcriptase fidelity and accuracy of RT–PCR procedures

In an effort to improve reverse transcriptase (RT) fidelity, we measured the error rate of Moloney murine leukemia virus (MMLV) RT in the presence of several autonomous and DNA polymerase-associated 3′–5′ exonucleases using a lacZ forward mutation assay. A number of 3′–5′ exonucleases were found to lower the error rate of MMLV RT, including p53, Escherichia coli DNA polymerase III ε subunit, and the proofreading activities associated with T4, ϕ29, and E. coli pol I DNA polymerases. The bacterial ε subunit increased RNA-dependent DNA synthesis fidelity by approximately threefold and was the only 3′-5′ exonuclease tested that did not deleteriously affect RT–PCR yields. Further testing showed that RT–PCR mutant frequencies were reduced significantly by performing cDNA synthesis in the presence of ε subunit, followed by PCR with a high-fidelity proofreading DNA polymerase. DNA sequence analysis was used to show that the combination of MMLV RT/ε subunit and PfuUltra DNA polymerase produces approximately eightfold fewer errors compared with the commonly used combination of MMLV RT and a Taq-based high-fidelity blend, consistent with predictions based on experimentally determined polymerase error rates.

Crystal structures of oligonucleotides including the integrase processing site of the Moloney murine leukemia virus

In the first step of retroviral integration, integrase cleaves the linear viral DNA within its long terminal repeat (LTR) immediately 3′ to the CA dinucleotide step, resulting in a reactive 3′ OH on one strand and a 5′ two base overhang on the complementary strand. In order to investigate the structural properties of the 3′ end processing site within the Moloney murine leukemia virus (MMLV) LTR d(TCTTTCATT), a host-guest crystallographic method was employed to determine the structures of four self-complementary 16 bp oligonucleotides including LTR sequences (underlined), d(TTTCATTGCAATGAAA), d(CTTTCATTAATGAAAG), d(TCTTTCATATGAAAGA) and d(CACAATGATCATTGTG), the guests, complexed with the N-terminal fragment of MMLV reverse transcriptase, the host. The structures of the LTR-containing oligonucleotides were compared to those of non-LTR oligonucleotides crystallized in the same lattice. Properties unique to the CA dinucleotide step within the LTR sequence, independent of its position from the end of the duplex, include a positive roll angle and negative slide value. This propensity for the CA dinucleotide step within the MMLV LTR sequence to adopt only positive roll angles is likely influenced by the more rigid, invariable 3′ and 5′ flanking TT dinucleotide steps and may be important for specific recognition and/or cleavage by the MMLV integrase.

Revealing domain structure through linker-scanning analysis of the murine leukemia virus (MuLV) RNase H and MuLV and human immunodeficiency virus type 1 integrase proteins.

Linker-scanning libraries were generated within the 3' terminus of the Moloney murine leukemia virus (M-MuLV) pol gene encoding the connection-RNase H domains of reverse transcriptase (RT) as well as the structurally related M-MuLV and human immunodeficiency virus type 1 (HIV-1) integrase (IN) proteins. Mutations within the M-MuLV proviral vectors were Tn7 based and resulted in 15-bp insertions. Mutations within an HIV-1 IN bacterial expression vector were based on Tn5 and resulted in 57-bp insertions. The effects of the insertions were examined in vivo (M-MuLV) and in vitro (HIV-1). A total of 178 individual M-MuLV constructs were analyzed; 40 in-frame insertions within RT connection-RNase H, 108 in-frame insertions within IN, 13 insertions encoding stop codons within RNase H, and 17 insertions encoding stop codons within IN. For HIV-1 IN, 56 mutants were analyzed. In both M-MuLV and HIV-1 IN, regions are identified which functionally tolerate multiple-linker insertions. For MuLV, these correspond to the RT-IN proteolytic junction, the junction between the IN core and C terminus, and the C terminus of IN. For HIV-1 IN, in addition to the junction between the IN core and C terminus and the C terminus of IN, insertions between the N terminus and core domains maintained integration and disintegration activity. Of the 40 in-frame insertions within the M-MuLV RT connection-RNase H domains, only the three C-terminal insertions mapping to the RT-IN proteolytic junction were viable. These results correlate with deletion studies mapping the domain and subdomain boundaries of RT and IN. Importantly, these genetic footprints provide a means to identify nonessential regions within RT and IN for targeted gene therapy applications.

O-25: FOXL2, a gene associated with premature ovarian failure, interacts with large tumor suppressor gene 1 (LATS1)

Blepharophimosis-Ptosis-Epicanthus Inversus (BPES) type I is an autosomal dominant disorder wherein patients have facial features with characteristic eyelid dysplasia co-inherited with infertility, specifically abnormal ovarian function leading to premature ovarian failure. Mutations in the gene encoding a novel forkhead transcription factor, Forkhead L2 (FOXL2), have been found in patients with BPES type I thus producing a putative truncated protein. Like other forkhead transcription factors, the activity of FOXL2 is likely regulated by interacting proteins. Thus, we set out to determine if FOXL2 interacts with other proteins that may be pivotal in ovarian function. Further, we evaluated expression of one of these interacting proteins. Experiment A yeast two-hybrid screen was conducted using an ovary cDNA library to identify proteins that interact with full-length FOXL2. The open reading frame of human FOXL2 cDNA was fused in-frame with the GAL4-binding domain into the pGBT9 yeast shuttle vector (Clontech, Palo Alto, CA). This vector was used to identify FOXL2-interacting proteins by screening 1.5 million transformants from a GAL4 activation domain-tagged ovarian fusion cDNA library prepared from rats. Yeast cells were co-transformed with pGBT9-FOXL2 and cDNAs from the ovarian library, and colonies were selected in plates deficient in tryptophan, leucine, and histidine but containing 30 mM 3-amino-1,2,4-triazole (Sigma, St. Louis, MO). Plasmids were isolated from positive colonies following transformation of Escherichia coli cells and then sequenced. Based on the Yeast 2 hybrid results, reverse transcriptase PCR was performed on ovaries at different stages of ovarian development for FOXL2 and one of the interacting proteins found through the yeast two-hybrid screen. Fetal (17.5 days postconception), immature (day 13 and 23), and adult (7 week) mouse ovaries were obtained. Total RNA from whole ovaries was extracted. Reverse transcription of total RNA from mouse ovaries was performed using oligo (dT)18 primer and recombinant Moloney-murine leukemia virus reverse transcriptase (Clontech). PCR amplification was performed. After PCR, the amplified product was subjected to electrophoresis on a 1.5% agarose gel stained with ethidium bromide. Large Tumor Suppressor 1 (LATS1) gene was found to interact strongly with full-length FOXL2. Subsequently, RT-PCR demonstrated the presence of the FOXL2 transcript in fetal (17.5 days postconception), immature (day 13 and 23), and adult (7 week) mouse ovaries. Further, RT-PCR demonstrated the expression of LATS1 at similar stages of development. Large Tumor Suppressor 1 (LATS1) gene, a putative serine/threonine kinase and tumor suppressor gene, was found to interact strongly with FOXL2. Mice null for the LATS1 gene have an ovarian phenotype reminiscent of premature ovarian failure. Our data demonstrates that LATS1 transcript is expressed at similar stages of development as FOXL2. Based on similar ovarian phenotypes with loss of function mutations in FOXL2 and LATS1 and expression of the FOXL2 and LATS1 transcript in similar stages of ovarian development, we propose they function through a similar pathway during follicle maintenance.

Evolution and Function of Leukocyte RNase A Ribonucleases of the Avian Species, Gallus gallus

In this study, we explore the evolution and function of two closely related RNase A ribonucleases from the chicken, Gallus gallus. Separated by approximately 10 kb on chromosome 6, the coding sequences of RNases A-1 and A-2 are diverging under positive selection pressure (dN > dS) but remain similar to one another (81% amino acid identity) and to the mammalian angiogenins. Immunoreactive RNases A-1 and A-2 (both approximately 16 kDa) were detected in peripheral blood granulocytes and bone marrow. Recombinant proteins are ribonucleolytically active (kcat = 2.6 and 0.056 s(-1), respectively), and surprisingly, both interact with human placental ribonuclease inhibitor. RNase A-2, the more cationic (pI 11.0), is both angiogenic and bactericidal; RNase A-1 (pI 10.2) has neither activity. We demonstrated via point mutation of the catalytic His110 that ablation of ribonuclease activity has no impact on the bactericidal activity of RNase A-2. We determined that the divergent domains II (amino acids 71-76) and III (amino acids 89-104) of RNase A-2 are both important for bactericidal activity. Furthermore, we demonstrated that these cationic domains can function as independent bactericidal peptides without the tertiary structure imposed by the RNase A backbone. These results suggest that ribonucleolytic activity may not be a crucial constraint limiting the ongoing evolution of this gene family and that the ribonuclease backbone may be merely serving as a scaffold to support the evolution of novel, nonribonucleolytic proteins.

Increased CUG Triplet Repeat-binding Protein-1 Predisposes to Impaired Adipogenesis with Aging

Preadipocyte differentiation capacity declines between middle and old age. Expression of the adipogenic transcription factors, CCAAT/enhancer-binding protein (C/EBP) alpha and peroxisome proliferator-activated receptor gamma (PPARgamma), is lower in differentiating preadipocytes from old than young animals, although no age-related changes occur in C/EBPbeta mRNA, which is upstream of C/EBPalpha and PPARgamma. C/EBPbeta-liver-enriched inhibitory protein (C/EBPbeta-LIP), a truncated C/EBPbeta isoform that is a dominant inhibitor of differentiation, increases with aging in rat fat tissue and preadipocytes. CUG triplet repeat-binding protein-1 (CUGBP1) binds to C/EBPbeta mRNA, increasing C/EBPbeta-LIP translation. Abundance and nucleotide binding activity of CUGBP1 increased with aging in preadipocytes. CUGBP1 overexpression in preadipocytes from young animals increased C/EBPbeta-LIP and impaired adipogenesis. Decreasing CUGBP1 in preadipocytes from old rats by RNA interference reduced C/EBPbeta-LIP abundance and promoted adipogenesis. Tumor necrosis factor-alpha, levels of which are elevated in fat tissue with aging, increased CUGBP1 protein, CUGBP1 binding activity, and C/EBPbeta-LIP in preadipocytes from young rats. Thus, CUGBP1 contributes to regulation of adipogenesis in primary preadipocytes and is responsive to tumor necrosis factor-alpha. With aging, preadipocyte CUGBP1 abundance and activity increases, resulting in enhanced translation of the C/EBPbeta-LIP isoform, thereby blocking effects of adipogenic transcription factors, predisposing preadipocytes from old animals to resist adipogenesis. Altered translational processing, possibly related to changes in cytokine milieu and activation of stress responses, may contribute to changes in progenitor differentiation and tissue function with aging.

The Biochemical Characterization of Ferret Carotene-9′, 10′-Monooxygenase Catalyzing Cleavage of Carotenoids in Vitro and in Vivo

Previous studies have shown that beta-carotene 15,15'-monooxygenase catalyzes the cleavage of beta-carotene at the central carbon 15,15'-double bond but cleaves lycopene with much lower activity. However, expressing the mouse carotene 9',10'-monooxygenase (CMO2) in beta-carotene/lycopene-synthesizing and -accumulating Escherichia coli strains leads to both a color shift and formation of apo-10'-carotenoids, suggesting the oxidative cleavage of both carotenoids at their 9',10'-double bond. Here we provide information on the biochemical characterization of CMO2 of the ferret, a model for human carotenoid metabolism, in terms of the kinetic analysis of beta-carotene/lycopene cleavage into beta-apo-10'-carotenal/apo-10'-lycopenal in vitro and the formation of apo-10'-lycopenoids in ferrets in vivo. We demonstrate that the recombinant ferret CMO2 catalyzes the excentric cleavage of both all-trans-beta-carotene and the 5-cis- and 13-cis-isomers of lycopene at the 9',10'-double bond but not all-trans-lycopene. The cleavage activity of ferret CMO2 was higher toward lycopene cis-isomers as compared with beta-carotene as substrate. Iron was an essential co-factor for the reaction. Furthermore, all-trans-lycopene supplementation in ferrets resulted in significant accumulation of cis-isomers of lycopene and the formation of apo-10'-lycopenol, as well as up-regulation of the CMO2 expression in lung tissues. In addition, in vitro incubation of apo-10'-lycopenal with the post-nuclear fraction of hepatic homogenates of ferrets resulted in the production of both apo-10'-lycopenoic acid and apo-10'-lycopenol, respectively, depending upon the presence of NAD+ or NADH as cofactors. Our finding of bioconversion of cis-isomers of lycopene into apo-10'-lycopenoids by CMO2 is significant because cis-isomers of lycopene are a predominant form of lycopene in mammalian tissues and apo-lycopenoids may have specific biological activities related to human health.

Rolling circle amplification-RACE: a method for simultaneous isolation of 5′ and 3′ cDNA ends from amplified cDNA templates

Isolation of full-length gene transcripts is important to determine the protein coding region and study gene structure. However, isolation of novel gene sequences is often limited to expressed sequence tags (ESTs) (i.e., short cDNA fragments that predominantly represent the 3′ end of the transcript). Rapid amplification of cDNA ends (RACE) is today by far the most popular approach for obtaining full-length cDNAs when only part of the transcript’s sequence is known.Since its original description (1,2) numerous modifications and improve-ments of the method have been developed and consist of a collection of PCR-based cloning procedures that extend a known cDNA fragment toward the 3′ (3′ RACE) or the 5′ (5′ RACE) cDNA end. The original method is based on attachment of an anchor sequence to one end of the cDNA that can be used as a primer binding template in PCR with a second gene-specific primer from the known part of the gene. Although this procedure seems in theory fast and simple, it is technically difficult and usually requires substantial optimi-zation and several repetitions before satisfactory results can be obtained (3). This is particularly due to the use of a universal primer corresponding to the anchor sequence present in all cDNAs, which may result in a high background of nonspecific products even after a nested PCR with a gene-specific primer internal to the first gene-specific primer is performed. Another drawback of the method is the difficulty of obtaining the full-length 5′ end of the transcript due to the presence of many truncated transcripts in the messenger RNA (mRNA) pool. Several strategies aimed at eliminating these problems have been developed (4–9) and have proven to be very useful in certain applica-tions. One improvement is based on the utilization of a pair of gene-specific primers in inverse PCR on circularized cDNA templates, which would avoid the use of a universal primer and the problems it may generate (4,6,8–10). This strategy also allows the simulta-neous isolation of both cDNA ends in a single reaction (9,10). Some of these procedures require the generation of double-stranded cDNA, including the use of template-switching reverse transcription (9) or a post-reverse transcription adaptor ligation step (10). Methods that are performed directly on first-strand cDNA are complicated by the low efficiency of RNA ligase for the circularization reaction (6) or the need for bridging oligonucleotides for this step (8). Furthermore, existing inverse-RACE methods typically require nested PCR to amplify the transcript of interest, and only a limited number of transcripts can be isolated from a single reverse transcription reaction, making it difficult to analyze rare transcripts from scarce tissue.We describe here an improved inverse-RACE method, which uses CircLigase™ (Epicentre Biotechnologies, Madison, WI, USA) for cDNA circularization, followed by rolling circle amplification (RCA) of the circular cDNA with ϕ29 DNA polymerase (New England Biolabs, Ipswich, MA, USA). In this way, a large amount of the PCR template is produced, allowing the simultaneous isolation of the 3′ and 5′ unknown ends of a virtually unlimited number of transcripts after a single reverse transcription reaction. Figure 1 illus-trates this method, named RCA-RACE. The process takes advantage of the properties of CircLigase to circularize

Specific Detection of Cytokeratin 20-Positive Cells in Blood of Colorectal and Breast Cancer Patients by a High Sensitivity Real-Time Reverse Transcriptase-Polymerase Chain Reaction Method

A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method for detection of cytokeratin 20-positive cells in blood characterized by two novel features was developed and tested on 99 patients with colorectal cancer, 110 with breast cancer, and 150 healthy subjects. To optimize the specificity and sensitivity of the method, two novel features were used. First, a primer overlapping two adjacent exons was generated to inhibit nonspecific amplification both in healthy donors and cancer patients; second, a non-end-point first-round amplification was used to increase sensitivity. The number of first-round cycles was chosen to reach the highest level of sensitivity while conserving quantitative characteristics. PCR efficiency increased from 88.9% in single-round RT-PCR to 99.0% in nested real-time RT-PCR. To establish sensitivity and specificity of the method, HT29 cells were serially diluted with normal blood. Detection limit improved from 100 HT29 cells (single-round RT-PCR) to 1 to 10 cells (nested real-time RT-PCR) per 3 ml of whole blood. None of the healthy subjects was positive, whereas 22 and 29% of all colorectal and breast cancer patients, respectively, had cytokeratin 20 cell equivalents in blood. The association between cytokeratin 20 cell equivalents and metastasis was statistically significant for breast (P = 0.026) but not colorectal cancer patients (P = 0.361). Negativity of all 150 healthy controls examined confers diagnostic potential to the method.