Strand-specific detection of overlapping transcripts via purification involving denaturation of biotinylated cDNA

Reverse transcription-PCR (RT-PCR) is the most widely employed technique for gene expression analysis owing to its high sensitivity, easy reproducibility and fast output. It has been conceived that priming RT reactions with gene-specific primers generates cDNA only from the specific RNA. However, several reports have revealed that cDNA is synthesized even without addition of exogenous primers in RT reactions. Owing to such self-priming activity, the signals from specific strands cannot be accurately detected and can confound the expression analysis, especially in context of overlapping bidirectional transcripts. Here, we demonstrate that purification of biotin-tagged cDNA in conjunction with alkaline denaturation can obviate the problem of background priming and enable accurate strand-specific detection of overlapping transcripts.

Coexistence of hemoglobin Handsworth and alpha 3.7 kb deletion in Caucasian woman in Poland

BackgroundThis report presents a case of an adult Polish women of Caucasian origin who was heterozygous for the nondeletional mutation: Hb Handsworth (HBA2 or HBA1: c.55G > C, p.Gly19Arg) and deletional (-α3.7) α-thalassemia mutation.MethodsThe HbA2 and HbF levels were measured by microcolumn chromatography and alkaline denaturation procedure, respectively, while electrophoresis was used to detect pathological hemoglobin fraction. The β- and α-globin genotypes were determined by DNA sequencing, gap-polymerase chain reaction, α gene triplication and MLPA.ResultsThe HbA2 and HbF levels were normal, but hemoglobin electrophoresis on agarose gel alkaline pH showed a strong band migration in a position of hemoglobin S and faint bands in the neighborhood of band A on acid electrophoresis. Molecular analysis of the alpha globin cluster detected a point mutation at codon 19 in HBA2 (c.55G > C, p.Gl- y19Arg) and deletion -α3.7.ConclusionsOur compound heterozygosity does not produce severe clinical or hematological symptoms but it is important to say that in our part of Europe such cases do appear. Molecular analysis of the alpha globin cluster is required for correct diagnosis in patients with normal HbA2 levels. Compound heterozygosity was unmasked by molecular diagnosis only.

Structural dissection of alkaline-denatured pepsin

Pepsin, a gastric aspartic proteinase, is a zymogen‒derived protein that undergoes irreversible alkaline denaturation at pH 6–7. Detailed knowledge of the structure of the alkaline‒denatured state is an important step in understanding the mechanism of the formation of the active enzyme. It has been established in a number of studies that the alkaline‒denatured state of pepsin (the IPstate) is composed of a compact C‒terminal lobe and a largely unstructured N‒terminal lobe. In the present study, we have investigated the residual structure in the IPstate in more detail, using limited proteolysis to isolate and characterize a tightly folded core region from this partially denatured pepsin. The isolated core region corresponds to the 141 C‒terminal residues of the pepsin molecule, which in the fully native state forms one of the two lobes of the structure. A comparative study using NMR and CD spectroscopy has revealed, however, that the N‒terminal lobe contributes a substantial amount of additional residual structure to the IPstate of pepsin. CD spectra indicate in addition that significant non‒native α-helical structure is present in the C‒terminal lobe of the structure when the N‒terminal lobe of pepsin is either unfolded or removed by proteolysis. This study demonstrates that the structure of pepsin in the IPstate is significantly more complex than that of a fully folded C‒terminal lobe connected to an unstructured N‒terminal lobe. The “misfolding” in this state could inhibit the proper refolding of the protein when returned to conditions that stabilize the native state.