Stem-loop Secondary Structure Research Articles

Complete mtDNA of Meretrix lusoria (Bivalvia: Veneridae) reveals the presence of an atp8 gene, length variation and heteroplasmy in the control region

The complete nucleotide sequence of the mitochondrial genome of the clam Meretrix lusoria (Bivalvia: Veneridae) was determined. It comprises 20,268 base pairs (bp) and contains 13 protein-coding genes, including ATPase subunit 8 ( atp8), two ribosomal RNAs, 22 transfer RNAs, and a non-coding control region. The atp8 encodes a protein of 39 amino acids. All genes are encoded on the same strand. A putative control region (CR or D-loop) was identified in the major non-coding region (NCR) between the tRNA Gly and tRNA Gln . A 1087 bp tandem repeat fragment was identified that comprises nearly 11 copies of a 101 bp motif and accounts for approximately 41% of the NCR. The 101 bp tandem repeat motif of the NCR can be folded into a stem–loop secondary structure. Samples of eight individuals from Hainan and Fujian provinces were collected and their NCR regions were successfully amplified and sequenced. The data revealed a highly polymorphic VNTR (variable number of tandem repeats) associated with high levels of heteroplasmy in the D-loop region. The size of the CR ranged from 1942 to 3354 bp depending upon the copy number of the repeat sequence.

A Comparative Analysis of Mitochondrial Genomes in Orthoptera (Arthropoda: lnsecta) and Genome Descriptions of Three Grasshopper Species

The complete sequences of mitochondrial DNA (mtDNA) from the three new grasshopper species, Euchorthippus fusigeniculatus, Mekongiana xiangchengensis and Mekongiella xizangensis, consisting of 15772 bp, 15567 bp, and 15885 bp, respectively, were analyzed and compared to mtDNAs from other 19 Orthoptera species obtained from GenBank. The three mitochondrial genomes contain a standard set of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes. and an A+T-rich region in the same order as those of the other analyzed caeliferan species, but different from those of the ensiferan species by the rearrangement of trnD and trnK. The putative initiation codon for the cox1 gene is ATC in E. fusigeniculatus, CTG in M. xiangchengensis and CCG in M. xizangensis. All secondary structures of tRNA-Ser((AGN)) in the three species lack a DHU arm. In this study, we stressed the comparative analysis of the stem-loop secondary structure in A+T-rich region of all Orthoptera species available to date, and report new findings which may facilitate further investigation and better understanding of this secondary structure. Finally, we undertook a phylogenetic study of all Orthoptera species available from GenBank to date based on three different datasets using parsimony, maximum likelihood, and Bayesian inference. Our result showed that protein-coding genes (PCG) and amino acid sequences (PCG_PROT) provided good resolution of higher-level relationships within the Orthoptera, whereas ribosomal RNA genes (RIBO) perform poorly under different optimality criteria.

New approach to real-time nucleic acids detection: folding polymerase chain reaction amplicons into a secondary structure to improve cleavage of Förster resonance energy transfer probes in 5′-nuclease assays

The article describes a new technology for real-time polymerase chain reaction (PCR) detection of nucleic acids. Similar to Taqman, this new method, named Snake, utilizes the 5′-nuclease activity of Thermus aquaticus (Taq) DNA polymerase that cleaves dual-labeled Förster resonance energy transfer (FRET) probes and generates a fluorescent signal during PCR. However, the mechanism of the probe cleavage in Snake is different. In this assay, PCR amplicons fold into stem–loop secondary structures. Hybridization of FRET probes to one of these structures leads to the formation of optimal substrates for the 5′-nuclease activity of Taq. The stem–loop structures in the Snake amplicons are introduced by the unique design of one of the PCR primers, which carries a special 5′-flap sequence. It was found that at a certain length of these 5′-flap sequences the folded Snake amplicons have very little, if any, effect on PCR yield but benefit many aspects of the detection process, particularly the signal productivity. Unlike Taqman, the Snake system favors the use of short FRET probes with improved fluorescence background. The head-to-head comparison study of Snake and Taqman revealed that these two technologies have more differences than similarities with respect to their responses to changes in PCR protocol, e.g. the variations in primer concentration, annealing time, PCR asymmetry. The optimal PCR protocol for Snake has been identified. The technology’s real-time performance was compared to a number of conventional assays including Taqman, 3′-MGB-Taqman, Molecular Beacon and Scorpion primers. The test trial showed that Snake supersedes the conventional assays in the signal productivity and detection of sequence variations as small as single nucleotide polymorphisms. Due to the assay’s cost-effectiveness and simplicity of design, the technology is anticipated to quickly replace all known conventional methods currently used for real-time nucleic acid detection.

Experimental evidence that RNA recombination occurs in the Japanese encephalitis virus

Due to the lack of a proofreading function and error-repairing ability of genomic RNA, accumulated mutations are known to be a force driving viral evolution in the genus Flavivirus, including the Japanese encephalitis (JE) virus. Based on sequencing data, RNA recombination was recently postulated to be another factor associated with genomic variations in these viruses. We herein provide experimental evidence to demonstrate the occurrence of RNA recombination in the JE virus using two local pure clones (T1P1-S1 and CJN-S1) respectively derived from the local strains, T1P1 and CJN. Based on results from a restriction fragment length polymorphism (RFLP) assay on the C/preM junction comprising a fragment of 868 nucleotides (nt 10-877), the recombinant progeny virus was primarily formed in BHK-21 cells that had been co-infected with the two clones used in this study. Nine of 20 recombinant forms of the JE virus had a crossover in the nt 123-323 region. Sequencing data derived from these recombinants revealed that no nucleotide deletion or insertion occurred in this region favoring crossovers, indicating that precisely, not aberrantly, homologous recombination was involved. With site-directed mutagenesis, three stem-loop secondary structures were destabilized and re-stabilized in sequence, leading to changes in the frequency of recombination. This suggests that the conformation, not the free energy, of the secondary structure is important in modulating RNA recombination of the virus. It was concluded that because RNA recombination generates genetic diversity in the JE virus, this must be considered particularly in studies of viral evolution, epidemiology, and possible vaccine safety.

The PKR-binding domain of adenovirus VA RNAI exists as a mixture of two functionally non-equivalent structures

VA RNAI is a non-coding adenoviral transcript that counteracts the host cell anti-viral defenses such as immune responses mediated via PKR. We investigated potential alternate secondary structure conformations within the PKR-binding domain of VA RNAI using site-directed mutagenesis, RNA UV-melting analysis and enzymatic RNA secondary structure probing. The latter data clearly indicated that the wild-type VA RNAI apical stem can adopt two different conformations and that it exists as a mixed population of these two structures. In contrast, in two sequence variants we designed to eliminate one of the possible structures, while leaving the other intact, each formed a unique secondary structure. This clarification of the apical stem pairing also suggests a small alteration to the apical stem–loop secondary structure. The relative ability of the two apical stem conformations to bind PKR and inhibit kinase activity was measured by isothermal titration calorimetry and PKR autophosphorylation inhibition assay. We found that the two sequence variants displayed markedly different activities, with one being a significantly poorer binder and inhibitor of PKR. Whether the presence of the VA RNAI conformation with reduced PKR inhibitory activity is directly beneficial to the virus in the cell for some other function requires further investigation.

Positional effect of mutations in 5'UTR of hepatitis C virus 4a on patients' response to therapy

To investigate the effects of mutations in domain III of the hepatitis C virus (HCV) internal ribosome entry sequences (IRES) on the response of chronic HCV genotype 4a patients to interferon therapy. HCV RNA was extracted from 19 chronic HCV 4a patients receiving interferon/ribavirin therapy who showed dramatic differences in their response to combination therapy after initial viral clearance. IRES domain III was cloned and 15 clones for each patient were sequenced. The obtained sequences were aligned with genotype 4a prototype using the ClustalW program and mutations scored. Prediction of stem-loop secondary structure and thermodynamic stability of the major quasispecies in each patient was performed using the MFOLD 3.2 program with Turner energies and selected constraints on base pairing. Analysis of RNA secondary structure revealed that insertions in domain III altered Watson-Crick base pairing of stems and reduced molecular stability of RNA, which may ultimately reduce binding affinity to ribosomal proteins. Insertion mutations in domain III were statistically more prevalent in sustained viral response patients (SVR, n = 14) as compared to breakthrough (BT, n = 5) patients. The influence of mutations within domain III on the response of HCV patients to combination therapy depends primarily on the position, but not the frequency, of these mutations within IRES domain III.

Massively Parallel High Throughput Sequencing Identifies Novel Micrornas in Normal and Malignant B Cells.

Background: MicroRNAs (miRNAs) are small non coding RNAs that have been shown to play a regulatory role in a number of different settings including development, hematopoiesis and lineage-selection. The expression patterns of miRNAs in various cellular processes and in various normal and malignant tissues are an area of active exploration. Bioinformatic analyses of the genome suggest that there might be thousands of miRNAs encoded in the genome. However, thus far only about 600 unique miRNAs have been identified in humans. The role of microRNAs in B cell malignancies is poorly understood. Mature B cells comprise naive, germinal center, memory and plasma cells. These B cell stages comprise the majority of leukemias and lymphomas. We have previously demonstrated a role for microRNAs in the regulation of key transcription factors and oncogenes including PRDM1, LMO2 and MYBL1. We hypothesized that microRNAs play a role in the pathogenesis of lymphomas and have applied high throughput sequencing to understand the pattern and function of microRNA expression in normal B cells and their malignant counterparts.Methods: CD19+ mature B cells were obtained from normal patients undergoing tonsillectomy and sorted using flow cytometry into naive, germinal center, memory and plasma cells. We also obtained cells from tumor cell lines derived from mantle cell lymphoma (Mino, JVM2), Burkitt lymphoma (Ramos, BL41) and multiple myeloma (H929, U266), as well as patient tumor samples derived from Burkitt lymphoma and diffuse large B cell lymphomas. From these cells, we purified small RNAs (18-25 nucleotides) and ligated sequencing adapters to these small RNAs and subjected them to 15 cycles of PCR amplification. The constructs were then subjected to massively parallel high throughput sequencing (Illumina) in picoliter wells to identify millions of sequences per sample. Sequences thus identified were matched to the genome and microRNAs were identified based on their characteristic stem-loop secondary structure, thermodynamic stability, and evidence of processing with the microRNA-related enzymes drosha and dicer.Results: Using massively parallel high-throughput sequencing of small RNAs isolated from these B cell subsets, we analyzed a total of 62,293,147 sequences (> 1.6 billion bases). We found that 261 known microRNAs are expressed in normal and malignant B cells, a number that is three times higher than previously recognized. Our work also identified the expression of 86 novel miRNAs in normal and malignant B cells, many of which appear to target genes important in B cell differentiation including BCL6, NLK, EBF, as well as oncogenes including MYC, LMO2, and CCND1. We found no evidence of decreased expression of microRNAs in B cell malignancies, in contrast to the described down-regulation of microRNAs in tumors from other lineages. On the other hand, there were striking differences between the microRNA expression patterns in normal and malignant B cells, although B cell malignancies still frequently express miRNAs that are characteristic of their normal B cell counterpart. Each malignancy had a characteristic pattern of microRNA expression that was distinct from other malignancies and normal B cells.Conclusion: Through high throughput sequencing, we have discovered novel microRNAs that target important oncogenes including BCL6, MYC, LMO2, and CCND1, suggesting a role for microRNAs in B cell lymphomas.

Toll-Like Receptor 9 Mediates CpG Oligonucleotide–Induced Cellular Invasion

Toll-like receptor 9 (TLR9) belongs to the innate immune system and recognizes microbial and vertebrate DNA. We showed previously that treatment with the TLR9-agonistic ODN M362 (a CpG sequence containing oligonucleotide) induces matrix metalloproteinase-13-mediated invasion in TLR9-expressing human cancer cell lines. Here, we further characterized the role of the TLR9 pathway in this process. We show that CpG oligonucleotides induce invasion in macrophages from wild-type C57/B6 and MyD88 knockout mice and in human MDA-MB-231 breast cancer cells lacking MyD88 expression. This effect was significantly inhibited in macrophages from TLR9 knockout mice and in human MDA-MB-231 breast cancer cells stably expressing TLR9 small interfering RNA or dominant-negative tumor necrosis factor receptor-associated factor 6 (TRAF6). Sequence modifications to the CpG oligonucleotides that targeted the stem loop and other secondary structures were shown to influence the invasion-inducing effect in MDA-MB-231 cells. In contrast, methylation of the cytosine residues of the parent CpG oligonucleotide did not affect the TLR9-mediated invasion compared with the unmethylated parent CpG oligonucleotide. Finally, expression of TLR9 was studied in clinical breast cancer samples and normal breast epithelium with immunohistochemistry. TLR9 staining localized in epithelial cells in both cancer and normal samples. The mean TLR9 staining intensity was significantly increased in the breast cancer cells compared with normal breast epithelial cells. In conclusion, our results suggest that TLR9 expression is increased in breast cancer and CpG oligonucleotide-induced cellular invasion is mediated via TLR9 and TRAF6, independent of MyD88. Further, our findings suggest that the structure and/or stability of DNA may influence the induction of TLR9-mediated invasion in breast cancer.

RNA silencing and HIV: A hypothesis for the etiology of the severe combined immunodeficiency induced by the virus

A novel intrinsic HIV-1 antisense gene was previously described with RNA initiating from the region of an HIV-1 antisense initiator promoter element (HIVaINR). The antisense RNA is exactly complementary to HIV-1 sense RNA and capable of forming ~400 base-pair (bp) duplex RNA in the region of the long terminal repeat (LTR) spanning the beginning portion of TAR in the repeat (R) region and extending through the U3 region. Duplex or double-stranded RNA of several hundred nucleotides in length is a key initiating element of RNA interference (RNAi) in several species. This HIVaINR antisense RNA is also capable of forming multiple stem-loop or hairpin-like secondary structures by M-fold analysis, with at least one that perfectly fits the criteria for a microRNA (miRNA) precursor. MicroRNAs (miRNAs) interact in a sequence-specific manner with target messenger RNAs (mRNAs) to induce either cleavage of the message or impede translation. Human mRNA targets of the predicted HIVaINR antisense RNA (HAA) microRNAs include mRNA for the human interleukin-2 receptor gamma chain (IL-2RG), also called the common gamma (γc) receptor chain, because it is an integral part of 6 receptors mediating interleukin signalling (IL-2R, IL-4R, IL-7R, IL-9R, IL-15R and IL-21R). Other potential human mRNA targets include interleukin-15 (IL-15) mRNA, the fragile × mental retardation protein (FMRP) mRNA, and the IL-1 receptor-associated kinase 1 (IRAK1) mRNA, amongst others. Thus the proposed intrinsic HIVaINR antisense RNA microRNAs (HAAmiRNAs) of the human immunodeficiency virus form complementary targets with mRNAs of a key human gene in adaptive immunity, the IL-2Rγc, in which genetic defects are known to cause an X-linked severe combined immunodeficiency syndrome (X-SCID), as well as mRNAs of genes important in innate immunity. A new model of intrinsic RNA silencing induced by the HIVaINR antisense RNA in the absence of Tat is proposed, with elements suggestive of both small interfering RNA (siRNA) and miRNA.

Nuclear Protein TIA-1 Regulates COL2A1 Alternative Splicing and Interacts with Precursor mRNA and Genomic DNA

The RNA-binding protein TIA-1 (T-cell-restricted intracellular antigen-1) functions in regulating post-transcriptional mechanisms, including precursor mRNA (pre-mRNA) alternative splicing and mRNA translation. Utilizing a mini-gene consisting of part of the type II procollagen gene (COL2A1), we show that TIA-1 interacts with a conserved AU-rich cis element in COL2A1 intron 2 and modulates alternative splicing of exon 2. This unique, highly conserved cis element containing stem-loop secondary structure was previously identified in our laboratory as an essential motif that controls the developmentally regulated exon 2 splicing switch during chondrogenesis (McAlinden, A., Havlioglu, N., Liang, L., Davies, S. R., and Sandell, L. J. (2005) J. Biol. Chem. 280, 32700-32711). In vivo binding of endogenous TIA-1 to the AU-rich cis element in COL2A1 pre-mRNA was confirmed by the ribonucleoprotein immunoprecipitation assay. Importantly, we also show that TIA-1 interacts with the equivalent DNA sequence with a preference for single-stranded rather than double-stranded DNA. Chromatin immunoprecipitation assays (including an additional RNase step) confirmed this interaction in vivo. Competition assays showed that TIA-1 apparently binds with higher affinity to DNA than to RNA. Finally, we show that this strong DNA-TIA-1 interaction can be disrupted by an RNA polymerase during active transcription. This suggests a potentially novel, dual role for TIA-1 in shuttling between DNA and RNA ligands to co-regulate COL2A1 expression at the level of transcription and pre-mRNA alternative splicing.

Comparative Analysis of Complete Mitochondrial DNA Control Region of Four Species of Strigiformes

The sequence of the whole mitochondrial (mt) DNA control region (CR) of four species of Strigiformes was obtained. Length of the CR was 3 290 bp, 2 848 bp, 2 444 bp, and 1 771 bp for Asio flammeus, Asio otus, Athene noctua, and Strix aluco, respectively. Interestingly, the length of the control region was maximum in Asio flammeus among all the avian mtDNA control regions sequenced thus far. In addition, the base composition and organization of mtDNA CR of Asio flammeus were identical to those reported for other birds. On the basis of the differential frequencies of base substitutions, the CR may be divided two variable domains, I and III, and a central conserved domain, II. The 3′ end of the CR contained many tandem repeats of varying lengths and repeat numbers. In Asio flammeus, the repeated sequences consisted of a 126 bp sequence that was repeated seven times and a 78 bp sequence that was repeated 14 times. In Asio otus, there were also two repeated sequences, namely a 127 bp sequence that was repeated eight times and a 78 bp sequence that was repeated six times. The control region of Athene noctua contained three sets of repeats: a 89 bp sequence that was repeated three times, a 77 bp sequence that was repeated four times, and a 71 bp sequence that was repeated six times. Strix aluco, however, had only one repeated sequence, a 78 bp sequence that was repeated five times. The results of this study seem to indicate that these tandem repeats may have resulted from slipped-strand mispairing during mtDNA replication. Moreover, there are many conserved motifs within the repeated units. These sequences could form stable stem-loop secondary structures, which suggests that these repeated sequences play an important role in regulating transcription and replication of the mitochondrial genome.

Structural organization of the mitochondrial DNA control region in Aedes aegypti

The complete A+T - rich region of Aedes aegypti mitochondrial DNA has been cloned and sequenced. In Argentinean populations of the species, a polymorphism in the length of the amplified fragment was observed. Nucleotide sequence comparison of the shortest and longest A+T - rich amplified fragments detected revealed the presence of 2 types of tandemly repeated blocks. The size variation observed in natural populations is mainly due to the presence of a variable number of a 181 bp tandem repeat unit, located toward the 12S rRNA gene end. The size of the longest A+T - rich region was of 2070 bp, representing the largest control sequence reported for any mosquito species. Few relevant short blocks of primary-sequence similarity conserved in the control region of mosquitoes and other insects were detected scattered throughout the whole region. Five putative stem-loop secondary structures were found, one of them flanked by conserved sequences described in other insects. Our results suggest that there are no universal models of structure-function relations in the control region of insect mtDNA. In addition, we identified a short A+T - rich variable segment in the Ae. aegyti control region that would be suitable for population genetic studies.

HIV-1 Tat Interactions with p300 and PCAF Transcriptional Coactivators Inhibit Histone Acetylation and Neurotrophin Signaling through CREB

The human immunodeficiency virus type-1 (HIV-1) infects microglia, macrophages, and astrocytes in the central nervous system (CNS) and may cause severe neurological diseases, such as AIDS-related dementias or progressive encephalopathies, as a result of CNS inflammation and neurotrophin signaling defects associated with expression of viral antigens and HIV-1 replication in the brain. The HIV Tat protein can be endocytosed by surrounding uninfected cells; interacts with transcriptional coactivators/acetyltransferases, p300/CREB-binding protein, and p300/CREB-binding protein-associated factor (PCAF); and induces neuronal apoptosis. Since nerve growth factor (NGF) receptor and brain-derived neurotrophic factor receptor signaling through CREB requires p300 and PCAF histone acetyltransferases, we sought to determine whether HIV-1 Tat coactivator interactions interfere with neurotrophin receptor signaling in neuronal cells. Here, we demonstrate that Tat-coactivator interactions inhibit NGF- and brain-derived neurotrophic factor-responsive CRE trans-activation and neurotrophin protection against apoptosis in PC12 and IMR-32 neuroblastoma cells. Purified recombinant Tat or Tat-derived synthetic peptides, spanning p300- and PCAF-binding sequences, inhibit histone H3/H4 acetylation in vitro. A Tat mutant, TatK28A/K50A, defective for binding p300 and PCAF, neither repressed NGF-responsive CRE transactivation nor inhibited histone acetylation. HIV-1 Tat interacts in PCAF complexes in post-mortem CNS tissues from donor neuro-AIDS patients, as determined by fluorescence resonance energy transfer immunoconfocal microscopy. Importantly, these findings suggest that HIV-1 Tat-coactivator interactions may contribute to neurotrophin signaling impairments and neuronal apoptosis associated with HIV-1 infections of the CNS.

The Topology of Bulges in the Long Stem of the Flavivirus 3′ Stem-Loop Is a Major Determinant of RNA Replication Competence

All flavivirus genomes contain a 3'terminal stem-loop secondary structure (3'SL) formed by the most downstream approximately 100 nucleotides (nt) of the viral RNA. The 3'SL is required for virus replication and has been shown to bind both virus-coded and cellular proteins. Results of the present study using an infectious DNA for WN virus strain 956 initially demonstrated that the dengue virus serotype 2 (DEN2) 3'SL nucleotide sequence could not substitute for that of the WN 3'SL to support WN genome replication. To determine what WN virus-specific 3'SL nucleotide sequences were required for WN virus replication, WN virus 3'SL nucleotide sequences were selectively deleted and replaced by analogous segments of the DEN2 3'SL nucleotide sequence such that the overall 3'SL secondary structure was not disrupted. Top and bottom portions of the WN virus 3'SL were defined according to previous studies (J. L. Blackwell and M. A. Brinton, J. Virol. 71:6433-6444, 1997; L. Zeng, L., B. Falgout, and L. Markoff, J. Virol. 72:7510-7522, 1998). A bulge in the top portion of the long stem of the WN 3'SL was essential for replication of mutant WN RNAs, and replication-defective RNAs failed to produce negative strands in transfected cells. Introduction of a second bulge into the bottom portion of the long stem of the wild-type WN 3'SL markedly enhanced the replication competence of WN virus in mosquito cells but had no effect on replication in mammalian cells. This second bulge was identified as a host cell-specific enhancer of flavivirus replication. Results suggested that bulges and their topological location within the long stem of the 3'SL are primary determinants of replication competence for flavivirus genomes.

Ilf3 and NF90 associate with the axonal targeting element of Tau mRNA.

In neurons, the selective translocation of Tau mRNA toward axons is due to the presence of a nucleotide sequence located in its 3' untranslated region and serving as axonal targeting element. Using this RNA sequence as a probe by a Northwestern approach, we have detected several proteins that interact with the targeting RNA element and could potentially be involved in Tau mRNA translocation, translation halting, and/or stabilization. Among them, two proteins were identified as the interleukin enhancer binding factor 3 (Ilf3) and NF90, two isoforms derived from a single gene product through alternative splicing. Each protein comprises two double-stranded RNA binding motifs that can interact with the predicted stem-loop secondary structure of the axonal targeting element. Specific antibodies raised against common or specific peptide sequences showed that both Ilf3 and NF90 are polymorphic proteins that are detected in neuronal nuclei and cell bodies, as well as in the proximal neuritic segments. This observation favors the idea that Ilf3 and NF90 are part of a protein complex that escorts Tau mRNA toward the axon.

The signal peptide sequence of a lytic transglycosylase of Neisseria meningitidis is involved in regulation of gene expression.

The 60 nucleotides encoding the signal peptide of the Neisseria meningitidis membrane-bound lytic transglycosylase (MltA) homologue GNA33 were found to exert a negative regulatory effect on expression of GNA33 from either a T7- or a P(lac)-driven system in Escherichia coli. Down-regulation was observed to occur at the transcriptional/post-transcriptional level and could possibly be ascribed to the formation of a stem-loop secondary structure within the signal peptide sequence. Slowing down the transcription rate through inhibition/titration of the RNA polymerase resulted in a considerable increase in mRNA accumulation, suggesting that a better coupling of translation to transcription would impede the formation of the putative secondary structure. Screening of synonymous mutations in the signal peptide sequence that showed high-level expression of an in-frame fusion to a reporter resulted in the isolation of several deletion mutants lacking most of the sequence participating in the putative secondary structure. Interestingly, the increase in the steady-state mRNA level observed in deletion mutants was higher, reaching a 300-fold increment, than that found in substitution mutants. Our results support the hypothesis that the rate of transcription controls the formation of a secondary structure in the region of the GNA33 transcript corresponding to the signal peptide sequence and this, when formed, negatively regulates expression.

Role of the Structure of the Top Half of HIV-1 cTAR DNA on the Nucleic Acid Destabilizing Activity of the Nucleocapsid Protein NCp7

The viral nucleic acid chaperone protein NCp7 of HIV-1 assists the two obligatory strand transfers required for the conversion of the genomic RNA into double-stranded DNA by reverse transcriptase. The first strand transfer necessitates the annealing of the early product of cDNA synthesis, the minus strand strong stop DNA (ss-cDNA) to the 3′ end of the genomic RNA. The hybridization reaction involves regions containing imperfect stem-loop (SL) structures, namely the TAR RNA at the 3′ end of the genomic RNA and the complementary sequence cTAR at the 3′ end of ss-cDNA. To pursue the characterization of the interaction between NCp7 and cTAR DNA, we investigated by absorbance, steady-state and time-resolved fluorescence spectroscopy, the interaction of NCp7 with wild-type and mutated DNAs representing the top half of cTAR. NCp7 was found to activate the transient melting of this cTAR DNA structure but less efficiently than that of cTAR lower half. The NCp7-induced destabilization of cTAR top half is dependent upon the three nucleotides bulging out of the stem, which thus represent a melting initiation site. In contrast, despite its ability to bind NCp7, the top loop does not play any significant role in NCp7-mediated melting. Thermodynamic data further suggest that NCp7-mediated destabilization of this cTAR structure correlates with the free energy changes afforded by destabilizing motifs like loops and bulges within the SL secondary structure. Interestingly, since NCp7 melts only short double-stranded sequences, destabilizing motifs need to be regularly positioned along the genomic sequence in order to promote strand transfer and thus genetic recombination during proviral DNA synthesis.

The complete mitochondrial genomes sequences of Asio flammeus and Asio otus and comparative analysis

The complete mitochondrial genomes of Asio flammeus and Asio otus were sequenced and found to span 18858 bp and 18493 bp, respectively. It is surprising to find the former to be the largest among all avian mitochondrial genomes sequenced so far. The two genomes have very similar gene order with that of Gallus gallus, neither contains the pseudo control region, but both have a single extra base, namely Cytidine, at position 174 in ND3 gene. The control regions of Asio flammeus and Asio otus' mitochondrial genomes span 3288 bp and 2926 bp respectively, which are the longest among vertebrates except for Myxine glutinosa and contribute to the large size of two genomes. The 3' end of the control region of Asio flammeus and Asio otus contains many tandemly repeated sequences, which are highly similar to a putative control element, i.e. Mt5, and may form stable stem-loop secondary structures. Such repeated sequences probably play an important role in regulating transcription and replication of mitochondrial genome. Our results may provide important clues for uncovering the origin and evolution mechanisms of mitochondrion genome.

Characterization of the RNA components of a putative molecular switch in the 3' untranslated region of the murine coronavirus genome.

RNA virus genomes contain cis-acting sequence and structural elements that participate in viral replication. We previously identified a bulged stem-loop secondary structure at the upstream end of the 3' untranslated region (3' UTR) of the genome of the coronavirus mouse hepatitis virus (MHV). This element, beginning immediately downstream of the nucleocapsid gene stop codon, was shown to be essential for virus replication. Other investigators discovered an adjacent downstream pseudoknot in the 3' UTR of the closely related bovine coronavirus (BCoV). This pseudoknot was also shown to be essential for replication, and it has a conserved counterpart in every group 1 and group 2 coronavirus. In MHV and BCoV, the bulged stem-loop and pseudoknot are, in part, mutually exclusive, because of the overlap of the last segment of the stem-loop and stem 1 of the pseudoknot. This led us to hypothesize that they form a molecular switch, possibly regulating a transition occurring during viral RNA synthesis. We have now performed an extensive genetic analysis of the two components of this proposed switch. Our results define essential and nonessential components of these structures and establish the limits to which essential parts of each element can be destabilized prior to loss of function. Most notably, we have confirmed the interrelationship of the two putative switch elements. Additionally, we have identified a pseudoknot loop insertion mutation that appears to point to a genetic interaction between the pseudoknot and a distant region of the genome.

A novel sequence-based approach to localize translocation breakpoints identifies the molecular basis of a t(4;22).

Low copy repeats (LCRs) located in 22q11.2, especially LCR-B, are susceptible to rearrangements associated with several relatively common constitutional disorders. These include DiGeorge syndrome, Velocardiofacial syndrome, Cat-eye syndrome and recurrent translocations of 22q11 including the constitutional t(11;22) and t(17;22). The presence of palindromic AT-rich repeats (PATRRs) within LCR-B of 22q11.2, as well as within the 11q23 and 17q11 regions, has suggested a palindrome-mediated, stem-loop mechanism for the generation of such recurring constitutional 22q11.2 translocations. The mechanism responsible for non-recurrent 22q11.2 rearrangements is presently unknown due to the extensive effort required for breakpoint cloning. Thus, we have developed a novel fluorescence in-situ hybridization and primed in-situ hybridization (PRINS) approach and rapidly localized the breakpoint of a non-recurrent 22q11.2 translocation, a t(4;22). Multiple primer pairs were designed from the sequence of a 200 kb, chromosome 4, breakpoint-spanning BAC to generate PRINS probes. Amplification of adjacent primer pairs, labeled in two colors, allowed us to narrow the 4q35.1 breakpoint to a 6.7 kb clonable region. Application of our improved PRINS protocol facilitated fine-mapping the translocation breakpoints within 4q35.1 and 22q11.2, and permitted rapid cloning and analysis of translocation junction fragments. To confirm the PRINS localization results, PCR mapping of t(4;22) somatic cell hybrid DNA was employed. Analysis of the breakpoints demonstrates the presence of a 554 bp palindromic sequence at the chromosome 4 breakpoint and a 22q11.2 location within the same PATRR as the recurrent t(11;22) and t(17;22). The sequence of this breakpoint further suggests that a stem-loop secondary structure mechanism is responsible for the formation of other, non-recurrent translocations involving LCR-B of 22q11.2.