Codon reading patterns in Drosophila melanogaster mitochondria based on their tRNA sequences: a unique wobble rule in animal mitochondria.
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Mitochondrial (mt) tRNA(Trp), tRNA(Ile), tRNA(Met), tRNA(Ser)GCU, tRNA(Asn)and tRNA(Lys)were purified from Drosophila melanogaster (fruit fly) and their nucleotide sequences were determined. tRNA(Lys)corresponding to both AAA and AAG lysine codons was found to contain the anticodon CUU, C34 at the wobble position being unmodified. tRNA(Met)corresponding to both AUA and AUG methionine codons was found to contain 5-formylcytidine (f(5)C) at the wobble position, although the extent of modification is partial. These results suggest that both C and f(5)C as the wobble bases at the anticodon first position (position 34) can recognize A at the codon third position (position 3) in the fruit fly mt translation system. tRNA(Ser)GCU corresponding to AGU, AGC and AGA serine codons was found to contain unmodified G at the anticodon wobble position, suggesting the utilization of an unconventional G34-A3 base pair during translation. When these tRNA anticodon sequences are compared with those of other animal counterparts, it is concluded that either unmodified C or G at the wobble position can recognize A at the codon third position and that modification from A to t(6)A at position 37, 3'-adjacent to the anticodon, seems to be important for tRNA possessing C34 to recognize A3 in the mRNA in the fruit fly mt translation system. (+info)
CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs.
(26/3208)
Here we present CapSelect as a novel experimental approach for the selective enrichment of full-length cDNAs in PCR-mediated analysis of mRNA sequences. The method combines the 5'-CAP-dependent addition of specifically three to four non-templated dCMP residues to the 3'-end of full-length cDNAs by reverse transcriptases in the presence of manganese and the controlled ribonucleotide tailing of cDNA ends by terminal deoxynucleotidyl transferase using rATP. By virtue of the generated terminal sequence motif (5'-dC(3-4)rA(3-4)), full-length cDNAs are selectively anchored to a double-stranded DNA adapter (with a dT(3-4)dG(3)3'-overhang) by T4 DNA ligase. The technique described is highly efficient, discriminates premature termination products and enriches full-length cDNAs. (+info)
Genetic and antigenic diversity among eastern equine encephalitis viruses from North, Central, and South America.
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Eastern equine encephalitis virus (EEEV), the sole species in the EEE antigenic complex, is divided into North and South American antigenic varieties based on hemagglutination inhibition tests. Here we describe serologic and phylogenetic analyses of representatives of these varieties, spanning the entire temporal and geographic range available. Nucleotide sequencing and phylogenetic analyses revealed additional genetic diversity within the South American variety; 3 major South/Central American lineages were identified including one represented by a single isolate from eastern Brazil, and 2 lineages with more widespread distributions in Central and South America. All North American isolates comprised a single, highly conserved lineage with strains grouped by the time of isolation and to some extent by location. An EEEV strain isolated during a 1996 equine outbreak in Tamaulipas State, Mexico was closely related to recent Texas isolates, suggesting southward EEEV transportation beyond the presumed enzootic range. Plaque reduction neutralization tests with representatives from the 4 major lineages indicated that each represents a distinct antigenic subtype. A taxonomic revision of the EEE complex is proposed. (+info)
Molecular mechanisms of coxsackievirus persistence in chronic inflammatory myopathy: viral RNA persists through formation of a double-stranded complex without associated genomic mutations or evolution.
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Enterovirus infection and persistence have been implicated in the pathogenesis of certain chronic muscle diseases. In vitro studies suggest that persistent enteroviruses mutate, evolving into forms that are less lytic and display altered tropism, but it is less clear whether these mechanisms operate in vivo. In this study, persistent coxsackievirus RNA from the muscle of mice afflicted with chronic inflammatory myopathy (CIM) was characterized and compared with RNA from a virus that had established a persistent infection of G8 mouse myoblasts for 30 passages in vitro. Competitive strand-specific reverse transcription-PCR and susceptibility to RNase I treatment revealed that plus- and minus-strand viral RNAs were present at nearly equivalent levels in muscle and that they persisted in a double-stranded conformation. All regions of the viral genome persisted and were amplified as a series of seven overlapping fragments. Restriction endonuclease fingerprinting coupled with sequencing indicated that there was no evolution of the viral genome associated with its persistence in muscle. This contrasted with the productive persistent infection that was established in myoblast cultures, where plus-strand RNA predominated and persistent virus developed distinct mutations. In vitro persistence proceeded by a carrier culture mechanism and was completely dependent on production of infectious virus, since persistent viral RNA was not detected in cultures subjected to antibody-mediated curing. These experiments demonstrate that persistence of coxsackievirus RNA in muscle is not facilitated by distinct genetic changes in the virus that give rise to replication-defective forms but occurs primarily through production of stable double-stranded RNA that is produced as the acute viral infection resolves. The data suggest a mechanism for coxsackievirus persistence in myofibers and perhaps other nondividing cells whereby cells that survive infection could harbor persistent viral RNA for extended times without producing detectable levels of infectious virus. (+info)
Altered gene expression in melanocytes exposed to 4-tertiary butyl phenol (4-TBP): upregulation of the A2b adenosine receptor 1.
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Exposure to phenolic agents contributes to the development of occupational vitiligo. Proposed as a causative factor for leukoderma in vivo, the para-substituted phenol 4-tertiary butyl phenol was chosen to investigate early cellular events responsible for selective disappearance of melanocytes from the epidermis of individuals sensitive to such agents. To this end, differential display of melanocyte mRNA isolated from three separate cultures was performed following a 12 h exposure of cells to 250 microM 4-tertiary butyl phenol or to vehicle alone. Fragments of cDNA representing differentially expressed messages were cloned and subsequently confirmed by reverse dot blotting. Alignment analysis revealed that the L30 ribosomal protein was upregulated by the treatment, potentially reflecting altered levels of protein synthesis in response to stress. In addition, a gene sequence upregulated following exposure to 4-tertiary butyl phenol was identified as the A2b receptor (a P1 receptor for adenosine). Differential expression of this gene was confirmed in an RNase protection assay. By reverse transcription-polymerase chain reaction, the gene was shown to be expressed in keratinocytes and fibroblasts as well. Flow cytometry confirmed differential expression in melanocytes and fibroblasts, but not in keratinocytes. Interestingly, it has been reported that P1 purinoceptor stimulation can induce apoptosis. This is in concordance with results reported elsewhere demonstrating induction of apoptosis by 4-tertiary butyl phenol in human melanocytes, as well as with morphologic changes observed in this study in cells exposed to 250 microM 4-tertiary butyl phenol for 72 h. In conclusion, differential display is useful to establish melanocyte components involved in the cellular response to phenolic agents. (+info)
Efficient rescue of infectious bursal disease virus from cloned cDNA: evidence for involvement of the 3'-terminal sequence in genome replication.
(30/3208)
To study the mechanism of replication of infectious bursal disease virus (IBDV), and to determine factors on the IBDV RNA which are involved in viral replication, we used cloned full-length cDNA of both the A- and B-segments to generate infectious IBDV. Infectious IBDV was rescued from plasmids that contained full-length IBDV cDNA behind a T7 promoter, by transfecting these plasmids into cells which were infected with a recombinant Fowlpox virus that expressed T7 RNA polymerase. By using the cDNA transfection system we evaluated the effect of the length of the 3' terminus of the A-segment plus strand of IBDV. Although wild-type IBDV predominantly contains four cytosines at the 3' terminus, no difference in virus yield was found when virus was rescued from cDNAs containing three to six adjacent cytosines. When the 3' terminus was shorter than three cytosines the efficiency to generate infectious IBDV from cDNA was reduced, but IBDV could still be recovered reproducibly. The rescued viruses from cDNAs containing 3'-terminal deletions appeared to have a restored 3'-terminal sequence. The missing nucleotides are probably restored by using complementary bases of a stem-loop structure as template. (+info)
Restoration of the 3' end of potyvirus RNA derived from Poly(A)-deficient infectious cDNA clones.
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Poly(A)-deficient full-length cDNA clones of clover yellow vein virus (ClYVV), a member of the genus Potyvirus, were found to be infectious when expressed from the CaMV 35S promoter. The poly(A) tail was replaced with different short sequences and the infectivities of the cDNA constructs were examined. Although the infectivity of the plasmid varied depending on the sequences introduced, all the constructs were infectious. In all cases, progeny viral RNAs from the cDNA clones had an authentic viral sequence at their 3' regions with poly(A) tails and the downstream nonviral sequences were completely lost. However, two minor mutations, a two-nucleotide deletion at the 3' end and a single-nucleotide addition at the second nucleotide position downstream of the poly(A) site, were also observed. The clones of the viral (-) strand RNAs had poly(U) tracts at their 5' ends, suggesting that their synthesis is primed by the poly(U) sequence. It furthermore suggests that the mutations were introduced during or after primary transcription from the cDNA and were maintained during authentic viral replication. Although the mechanism involved is not known, recovery of the poly(A) tail is an essential step for maintaining the infectivity of the viral cDNAs. (+info)
Asymptomatic urethritis and detection of HIV-1 RNA in seminal plasma.
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OBJECTIVE: To define risk factors for detection of HIV-1 RNA in semen in men attending the two largest HIV clinics in the West Midlands. METHODS: 94 HIV-1 seropositive men at any stage of infection donated matched semen and blood samples. 36 subjects (38%) were on no antiretroviral treatment, 12 (13%) were on dual therapy, and 46 (49%) were on three or more drugs. Median CD4 count was 291 cells x 10(6)/l. 87 subjects underwent a urethritis screen (Gram stained urethral smear and culture for gonococcus, and LCR for Chlamydia trachomatis on first pass urine). Quantitative cell free HIV-1 RNA was determined by commercial nucleic acid sequence based assay with a lower detection limit of 800 copies/ml for semen and 400 copies/ml for blood. Independent risk factors for seminal HIV RNA detection were defined by logistic regression. RESULTS: In univariate analysis, subjects not taking antiretrovirals were 11 times more likely to shed HIV RNA (21/36 (58%) v 6/58 (10%); p < 0.0001). Seven subjects (8%) had urethritis (including one C trachomatis infection). Urethritis was significantly associated with detection of seminal HIV RNA (adjusted OR, 80.2; p = 0.006), as was blood plasma viral load (adj OR, 19.3 per factor 10 increase; p < 0.001) and age (adj OR, 1.16 per 1 year older; p = 0.001). Antiviral treatment status, absolute CD4 and CD8 count, clinical stage, treatment centre, ethnicity, and risk factor were not independent predictors. No subject with undetectable blood viral load had detectable seminal HIV RNA. CONCLUSION: Asymptomatic urethritis is independently associated with seminal HIV RNA shedding. (+info)