Targeted rapid amplification of cDNA ends (T-RACE)—an improved RACE reaction through degradation of non-target sequences

Amplification of the 5′ ends of cDNA, although simple in theory, can often be difficult to achieve. We describe a novel method for the specific amplification of cDNA ends. An oligo-dT adapter incorporating a dUTP-containing PCR primer primes first-strand cDNA synthesis incorporating dUTP. Using the Cap finder approach, another distinct dUTP containing adapter is added to the 3′ end of the newly synthesized cDNA. Second-strand synthesis incorporating dUTP is achieved by PCR, using dUTP-containing primers complimentary to the adapter sequences incorporated in the cDNA ends. The double-stranded cDNA-containing dUTP serves as a universal template for the specific amplification of the 3′ or 5′ end of any gene. To amplify the ends of cDNA, asymmetric PCR is performed using a single gene-specific primer and standard dNTPs. The asymmetric PCR product is purified and non-target transcripts containing dUTP degraded by Uracil DNA glycosylase, leaving only those transcripts produced during the asymmetric PCR. Subsequent PCR using a nested gene-specific primer and the 3′ or 5′ T-RACE primer results in specific amplification of cDNA ends. This method can be used to specifically amplify the 3′ and 5′ ends of numerous cDNAs from a single cDNA synthesis reaction

A non-canonical melanin biosynthesis pathway protects Aspergillus terreus conidia from environmental stress

Pro- and eukaryotes produce melanin for protection from environmental stress or as virulence determinant. The human pathogenic fungus Aspergillus fumigatus and related Ascomycetes produce dihydroxynaphthalene (DHN) melanin in conidia, which is essential for inhibiting phagolysosome acidification. In contrast, Aspergillus terreus lacks genes for biosynthesis of DHN-melanin. Therefore, the origin of the pigment in A. terreus conidia was elucidated. Expression analyses from conidiation conditions identified genes coding for an unusual NRPS-like enzyme (MelA) and a tyrosinase. MelA produces aspulvinone E as precursor, which is activated for polymerisation by the tyrosinase TyrP as shown by heterologous in vivo and in vitro reconstitution of pigment formation. Functional studies revealed that the pigment confers resistance against UV-light and hampers phagocytosis by soil amoeba, but does not inhibit acidification of phagolysosomes. Since A. terreus conidia prefer persistence at acidic pH, this uncommon type of melanin, termed Asp-melanin, might specifically contribute to survival in the environment

Discovery and Genomic Characterization of Noroviruses from a Gastroenteritis Outbreak in Domestic Cats in the US

Norovirus (NoV) RNA was detected in the stools of 6 out 14 (42.8%) 8–12-week-old cats with enteritis from a feline shelter, in New York State. Upon sequence analysis of the complete capsid, the six NoVs were found to be identical, suggesting the spread of a unique NoV strain in the shelter. The full-length genomic sequence (7839 nt) of one feline NoV, CU081210E/2010/US, was determined. In the capsid protein VP1 region, the virus displayed the highest amino acid identity to animal genogroup IV genotype 2 (GIV.2) NoVs: lion/Pistoia-387/06/IT (97.9%) and dog/Bari-170/07/IT (90.4%). These findings document the discovery of a novel feline calicivirus, different from vesiviruses, and extend the spectrum of NoV host range. Epidemiological studies using feline NoV-specific diagnostic tools and experimental infection of cats are required to understand whether NoVs have a pathogenic role in this species

Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes

We have developed an efficient strategy for cloning of PCR products that contain an unknown region flanked by a known sequence. As with ligation-independent cloning, the strategy is based on homology between sequences present in both the vector and the insert. However, in contrast to ligation-independent cloning, the cloning vector has homology with only one of the two primers used for amplification of the insert. The other side of the linearized cloning vector has homology with a sequence present in the insert, but nested and non-overlapping with the gene-specific primer used for amplification. Since only specific products contain this sequence, but none of the non-specific products, only specific products can be cloned. Cloning is performed using a one-step reaction that only requires incubation for 10 minutes at room temperature in the presence of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. The reaction mix is then directly transformed into E. coli where the annealed vector-insert complex is repaired and ligated. We have tested this method, which we call quick and clean cloning (QC cloning), for cloning of the variable regions of immunoglobulins expressed in non-Hodgkin lymphoma tumor samples. This method can also be applied to identify the flanking sequence of DNA elements such as T-DNA or transposon insertions, or be used for cloning of any PCR product with high specificity

Novel functions and regulation of cryptic cellobiose operons in Escherichia coli

Presence of cellobiose as a sole carbon source induces mutations in the chb and asc operons of Escherichia coli and allows it to grow on cellobiose. We previously engineered these two operons with synthetic constitutive promoters and achieved efficient cellobiose metabolism through adaptive evolution. In this study, we characterized two mutations observed in the efficient cellobiose metabolizing strain: duplication of RBS of ascB gene, (beta-glucosidase of asc operon) and nonsense mutation in yebK, (an uncharacterized transcription factor). Mutations in yebK play a dominant role by modulating the length of lag phase, relative to the growth rate of the strain when transferred from a rich medium to minimal cellobiose medium. Mutations in ascB, on the other hand, are specific for cellobiose and help in enhancing the specific growth rate. Taken together, our results show that ascB of the asc operon is controlled by an internal putative promoter in addition to the native cryptic promoter, and the transcription factor yebK helps to remodel the host physiology for cellobiose metabolism. While previous studies characterized the stress-induced mutations that allowed growth on cellobiose, here, we characterize the adaptation-induced mutations that help in enhancing cellobiose metabolic ability. This study will shed new light on the regulatory changes and factors that are needed for the functional coupling of the host physiology to the activated cryptic cellobiose metabolismopen1

The role of genomic location and flanking 3'UTR in the generation of functional levels of variant surface glycoprotein in Trypanosoma brucei

Trypanosoma brucei faces relentless immune attack in the mammalian bloodstream, where it is protected by an essential coat of Variant Surface Glycoprotein (VSG) comprising ∼10% total protein. The active VSG gene is in a Pol I-transcribed telomeric expression site (ES). We investigated factors mediating these extremely high levels of VSG expression by inserting ectopic VSG117 into VSG221 expressing T. brucei. Mutational analysis of the ectopic VSG 3′UTR demonstrated the essentiality of a conserved 16-mer for mRNA stability. Expressing ectopic VSG117 from different genomic locations showed that functional VSG levels could be produced from a gene 60 kb upstream of its normal telomeric location. High, but very heterogeneous levels of VSG117 were obtained from the Pol I-transcribed rDNA. Blocking VSG synthesis normally triggers a precise precytokinesis cell-cycle checkpoint. VSG117 expression from the rDNA was not adequate for functional complementation, and the stalled cells arrested prior to cytokinesis. However, VSG levels were not consistently low enough to trigger a characteristic ‘VSG synthesis block’ cell-cycle checkpoint, as some cells reinitiated S phase. This demonstrates the essentiality of a Pol I-transcribed ES, as well as conserved VSG 3′UTR 16-mer sequences for the generation of functional levels of VSG expression in bloodstream form T. brucei