Effect of mutations in a simian virus 40 PolyA signal enhancer on green fluorescent protein reporter gene expression.

Our previous studies have shown that tandem Alu repeats inhibit green fluorescent protein (GFP) gene expression when inserted downstream of the GFP gene in the pEGFP-C1 vector. We found that the 22R sequence (5'-GTGAAAAAAATGCTTTATTTGT-3') from the antisense PolyA (240 bp polyadenylation signal) of simian virus 40, eliminated repression of GFP gene expression when inserted between the GFP gene and the Alu repeats. The 22R sequence contains an imperfect palindrome; based on RNA structure software prediction, it forms an unstable stem-loop structure, including a loop, a first stem, a bulge, and a second stem. Analysis of mutations of the loop length of the 22R sequence showed that the three-nucleotide loop (wild-type, 22R) induced much stronger GFP expression than did other loop lengths. Two mutations, 4TMI (A7→T, A17→T) and 5AMI (A6→T, T18→A), which caused the base type changes in the bulge and in the second stem in the 22R sequence, induced stronger GFP gene expression than 22R itself. Mutation of the bulge base (A17→T), leading to complete complementation of the stem, caused weaker GFP gene expression. Sequences without a palindrome (7pieA, 5'-GTGAAAAAAATG CAAAAAAAGT-3', 7pieT, 5'-GTGTTTTTTTTGCTTTTTTTGT-3') did not activate GFP gene expression. We conclude that an imperfect palindrome affects and can increase GFP gene expression.

Multiple RNAs from the mouse carboxypeptidase M locus: functional RNAs or transcription noise?

BACKGROUND: A major effort of the scientific community has been to obtain complete pictures of the genomes of many organisms. This has been accomplished mainly by annotation of structural and functional elements in the genome sequence, a process that has been centred in the gene concept and, as a consequence, biased toward protein coding sequences. Recently, the explosion of transcriptome data generated and the discovery of many functional non-protein coding RNAs have painted a more detailed and complex scenario for the genome. Here we analyzed the mouse carboxypeptidase M locus in this broader perspective in order to define the mouse CPM gene structure and evaluate the existence of other transcripts from the same genomic region. RESULTS: Bioinformatic analysis of nucleotide sequences that map to the mouse CPM locus suggests that, in addition to the mouse CPM mRNA, it expresses at least 33 different transcripts, many of which seem to be non-coding RNAs. We randomly chose to evaluate experimentally four of these extra transcripts. They are expressed in a tissue specific manner, indicating that they are not artefacts or transcriptional noise. Furthermore, one of these four extra transcripts shows expression patterns that differed considerably from the other ones and from the mouse CPM gene, suggesting that there may be more than one transcriptional unit in this locus. In addition, we have confirmed the mouse CPM gene RefSeq sequence by rapid amplification of cDNA ends (RACE) and directional cloning. CONCLUSION: This study supports the recent view that the majority of the genome is transcribed and that many of the resulting transcripts seem to be non-coding RNAs from introns of genes or from independent transcriptional units. Although some of the information on the transcriptome of many organisms may actually be artefacts or transcriptional noise, we argue that it can be experimentally evaluated and used to find and define biological functional elements on the genome. Furthermore, the transcription of other functional RNAs besides the protein coding RNA from a specific genomic locus imposes extra care when designing and interpreting experiments involving genetic manipulations or expression detection and quantification.

Analysis of the genome of Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV-19) and of the high genomic heterogeneity in group II nucleopolyhedroviruses.

The genome of the most virulent among 22 Brazilian geographical isolates of Spodoptera frugiperda nucleopolyhedrovirus, isolate 19 (SfMNPV-19), was completely sequenced and shown to comprise 132,565 bp and 141 open reading frames (ORFs). A total of 11 ORFs with no homology to genes in the GenBank database were found. Of those, four had typical baculovirus promoter motifs and polyadenylation sites. Computer-simulated restriction enzyme cleavage patterns of SfMNPV-19 were compared with published physical maps of other SfMNPV isolates. Differences were observed in terms of the restriction profiles and genome size. Comparison of SfMNPV-19 with the sequence of the SfMNPV isolate 3AP2 indicated that they differed due to a 1427 bp deletion, as well as by a series of smaller deletions and point mutations. The majority of genes of SfMNPV-19 were conserved in the closely related Spodoptera exigua NPV (SeMNPV) and Agrotis segetum NPV (AgseMNPV-A), but a few regions experienced major changes and rearrangements. Synthenic maps for the genomes of group II NPVs revealed that gene collinearity was observed only within certain clusters. Analysis of the dynamics of gene gain and loss along the phylogenetic tree of the NPVs showed that group II had only five defining genes and supported the hypothesis that these viruses form ten highly divergent ancient lineages. Crucially, more than 60 % of the gene gain events followed a power-law relation to genetic distance among baculoviruses, indicative of temporal organization in the gene accretion process.

Expression of TPK1 and TPK2 genes encoding PKA catalytic subunits during growth and morphogenesis in Candida albicans.

The transcript levels of Candida albicans TPK1 and TPK2 genes, encoding PKA catalytic subunits, as well as phosphotransferase activity, were measured in the parental strain CAI4 and in homozygous tpk1Delta and tpk2Delta mutants during vegetative growth and during yeast-to-mycelial transition in N-acetylglucosamine liquid inducing medium at 37 degrees C. We observed two TPK2 transcripts, a major one of 1.8 kb and a minor one of 1.4 kb, and established by 3'-RACE that they originate from the recognition of the three polyadenylation signals present in the 3' untranslated region of the gene. During vegetative growth of CAI4 strain, the expression profiles of TPK1 and TPK2 varied similarly, reaching maximal expression at the late logarithmic phase. TPK1 mRNA levels were lower than those of TPK2 at all stages measured. In the corresponding homozygous tpk mutants, mRNA levels and the expression patterns of TPK1 and TPK2 were similar to those of CAI4, suggesting that the loss of one catalytic isoform is not compensated by overexpression of the other. Changes in PKA specific activity roughly correlated with fluctuations of mRNA expression levels. During yeast-to-mycelial transition, a sharp increase in TPK1 mRNA levels and in PKA-specific activity correlated with the onset of germ-tube formation in strain tpk2Delta. We also showed that tpk1Delta strain exhibited a delayed morphogenetic shift in comparison with CAI4 and tpk2Delta strains in several liquid inducing media, reinforcing the idea that Tpk1p is important for faster germ-tube appearance.

Molecular cloning and characterization of a cDNA encoding the Paracoccidioides brasiliensis 135 ribosomal protein.

A 630 bp cDNA encoding an L35 ribosomal protein of Paracoccidioides brasiliensis, designated as Pbl35, was cloned from a yeast expression library. Pbl35 encodes a polypeptide of 125 amino acids, with a predicted molecular mass of 14.5 kDa and a pI of 11.0. The deduced PbL35 shows significant conservation in respect to other described ribosomal L35 proteins from eukaryotes and prokaryotes. Motifs of ribosomal proteins are present in PbL35, including a bipartite nuclear localization signal (NLS) that could be related to the protein addressing to the nucleolus for the ribosomal assembly. The mRNA for PbL35, about 700 nucleotides in length, is expressed at a high level in P. brasiliensis. The PbL35 and the deduced amino acid sequence constitute the first description of a ribosomal protein in P. brasiliensis. The cDNA was deposited in GenBank under accession number AF416509.

Molecular cloning and characterization of a cDNA encoding the N-acetyl-beta-D-glucosaminidase homologue of Paracoccidioides brasiliensis.

A cDNA encoding the N-acetyl-beta-D-glucosaminidase (NAG) protein of Paracoccidioides brasiliensis, Pb NAG1, was cloned and characterized. The 2663-nucleotide sequence of the cDNA consisted of a single open reading frame encoding a protein with a predicted molecular mass of 64.73 kDa and an isoeletric point of 6.35. The predicted protein includes a putative 30-amino-acid signal peptide. The protein as a whole shares considerable sequence similarity with 'classic' NAG. The primary sequence of Pb NAG1 was used to infer phylogenetic relationships. The amino acid sequence of Pb NAG1 has 45, 31 and 30% identity, respectively, with homologous sequences from Trichoderma harzianum, Aspergillus nidulans and Candida albicans. In particular, striking homology was observed with the active site regions of the glycosyl hydrolase group of proteins (family 20). The expected active site consensus motif G X D E and catalytic Asp and Glu residues at positions 373 and 374 were found, reinforcing that Pb NAG1 belongs to glycosyl hydrolase family 20. The nucleotide sequence of Pb nag1 and its flanking regions have been deposited, along with the amino acid sequence of the deduced protein, in GenBank under accession number AF419158.

Sequence analysis of a 5.1 kbp region of the Spodoptera frugiperda multicapsid nucleopolyhedrovirus genome that comprises a functional ecdysteroid UDP-glucosyltransferase (egt) gene.

The ecdysteroid UDP-glucosyltransferase gene from the Spodoptera frugiperda multicapsid nucleopolyhedrovirus (SfMNPV) was identified using degenerate primers whose sequence were derived from conserved regions of the EGT proteins encoded by other baculoviruses. Analysis of the gene sequence revealed the presence of an open reading frame (ORF) with potential to encode a polypeptide of 525 amino acids. Promoter sequences typical of baculovirus genes were found in the 5' region of this ORF. A polyadenylation signal was identified downstream the translation stop codon. A transient expression assay showed that the product of this ORF was able to conjugate glucose from UDP-glucose with ecdysone confirming that the gene identified was indeed the SfMNPV egt gene. The SfMNPV egt gene and the sequences of other baculovirus egt genes were used to infer a phylogenetic tree. The nucleotide sequence of the entire BamHI fragment that contains the SfMNPV egt gene was determined. Search of the available sequence databases suggested that, besides the egt gene, this region contains 5 ORFs similar to the baculovirus genes gp37 (fusolin), to ptp2 and to ORFs 28, 29, and 30 of Spodoptera exigua multicapsid nucleopolyhedrovirus. Both the phylogenetic analysis of the egt genes and the gene order of the region that flanks the egt gene indicated that SfMNPV is closely related to the baculoviruses that infects S. exigua and Mamestra configurata.