Use of degenerate primers and touchdown PCR for construction of cDNA libraries

Optimized construction of low-redundancy cDNA mini-libraries using low-stringency RT-PCR is described cDNAs are generated using arbitrary consensus-degenerate hybrid oligonucleotide primers and nanogram amounts of Schistosoma mansoni mRNA. A number of conditions such as temperature and salt concentration are combined to create balanced, low-stringency conditions that permit a normalized amplification of a diversified, random set of sequences. On average, 350 different sequences are obtained per mini-library, which represents a significantly higher diversity of messages per library when compared to previously published conditions (ie., 20-40 sequences/ mini-library). The optimized high-throughput approach described here is likely to help in the discovery of expressed genes in any complex organism.     

Structure-function analysis of Escherichia coli DNA helicase I reveals non-overlapping transesterase and helicase domains

TraI (DNA helicase I) is an Escherichia coli F plasmid-encoded protein required for bacterial conjugative DNA transfer. The protein is a sequence-specific DNA transesterase that provides the site- and strand-specific nick required to initiate DNA strand transfer and a 5' to 3' DNA helicase that unwinds the F plasmid to provide the single-stranded DNA that is transferred from donor to recipient. Sequence comparisons with other transesterases and helicases suggest that these activities reside in the N- and C-terminal regions of TraI, respectively. Computer-assisted secondary structure probability analysis identified a potential interdomain region spanning residues 304-309. Proteins encoded by segments of traI, whose N or C terminus either flanked or coincided with this region, were purified and assessed for catalytic activity. Amino acids 1-306 contain the transesterase activity, whereas amino acids 309-1504 contain the helicase activity. The C-terminal 252 amino acids of the 1756-amino acid TraI protein are not required for either helicase or transesterase activity. Protein and nucleic acid sequence similarity searches indicate that the occurrence of both transesterase- and helicase-associated motifs in a conjugative DNA transfer initiator protein is rare. Only two examples (other than R100 plasmid TraI) were found: R388 plasmid TrwC and R46 plasmid (pKM101) TraH, belonging to the IncW and IncN groups of broad host range conjugative plasmids, respectively. The most significant structural difference between these proteins and TraI is that TraI contains an additional region of approximately 650 residues between the transesterase domain and the helicase-associated motifs. This region is required for helicase activity.     

Prediction of human protein function from post-translational modifications and localization features

We have developed an entirely sequence-based method that identifies and integrates relevant features that can be used to assign proteins of unknown function to functional classes, and enzyme categories for enzymes. We show that strategies for the elucidation of protein function may benefit from a number of functional attributes that are more directly related to the linear sequence of amino acids, and hence easier to predict, than protein structure. These attributes include features associated with post-translational modifications and protein sorting, but also much simpler aspects such as the length, isoelectric point and composition of the polypeptide chain.     

A new species,Litomosoides odilae n. sp (Nematoda: Onchocercidae) from Oligoryzomys nigripes (Rodentia: Muridae) in the rainforest of Misiones,Argentina

A new species of Litomosoides was collected from the abdominal cavity of Oligoryzomys nigripes (Rodentia: Muridae) in a semideciduous secondary rainforest of Misiones, Argentina. Litomosoides odilae n. sp. belongs to the carinii group and is characterized by the amphids displaced dorsally; buccal capsule with an anterior segment transparent and an annular asymmetrical thickening; esophagus divided, with the posterior glandular portion slightly wider than the muscular; male cloacal aperture strongly protruded; and microfilaria sheathed with an attenuated tail. The morphology of the new species, which is similar to that of L petteri, a parasite of marsupials in Brazil, suggests that host-switching events may have occurred in the diversification of this genus.     

The lower trapezius musculocutaneous flap revisited: versatile coverage for complicated wounds to the posterior cervical and occipital regions based on the deep branch of the transverse cervical artery

The clinical role of the lower trapezius musculocutaneous flap varies within the literature. Many describe its use in the reconstruction of the lateral neck and facial regions, but very few refer to its use in the posterior cervical and occipital regions. Different vascular pedicles have also been described and effectively used. A retrospective analysis was conducted, reviewing the authors' experience with 13 patients who suffered complex open wounds to the posterior cervical and occipital regions that were treated with a lower trapezius muscle or musculocutaneous flap. All flaps were based on the deep branch of the transverse cervical artery. This pedicle was used to support a relatively large skin segment over the distal portion of the lower trapezius muscle, a margin that, in the authors' experience, extends at least 1 cm beyond the muscular margin. Postoperatively, patients were evaluated based on complications, residual shoulder function, and aesthetic outcome. In addition to the clinical study, cadaveric dissection of the trapezius muscle was conducted on 22 specimens, and the vascular anatomy was confirmed by direct visualization. The authors' experience indicates that the lower trapezius musculocutaneous flap, when based on the deep branch of the transverse cervical artery, provides a reliable alternative for the reconstruction of complicated wounds in the posterior cervical and occipital regions, with the added capability of providing richly vascularized tissue to compromised wounds as far cephalad as the vertex of the skull.     

Characterization of Paracoccidioides brasiliensis atypical isolates by random amplified polymorphic DNA analysis

Two atypical Paracoccidioides brasiliensis strains (yeast form at room temperature) have been isolated from chronically infected patients living in Brazil. Different random primers were used to characterize these isolates and compare them to typical strains. The RAPD patterns allowed the differentiation of all the selected isolates. Their genetic distance ranged from 5% to 80% of non-shared bands depending on the strains and the primer used. The RAPD data were used to build a Wagner phenogram, which showed two major branched with more than 56% of genetic distance separating them. No significant difference was observed between the atypical isolates and the others suggesting that specific genes are involved in the dimorphism phenomenon.