ABSTRACT
BACKGROUND: DAZ is a male infertility gene located at the AZFc region of the Y chromosome. There are four copies of the DAZ gene that share a strong homology but are not identical to one another. In the present study, we carried out cDNA cloning and immunoblot analyses to determine whether all of the DAZ genes are actively expressed in the human testis. METHODS: AZFc deletion was detected by sequence-tagged site polymerase chain reaction (PCR) of genomic DNA isolated from blood samples. DAZ cDNAs were cloned with RT-PCR followed by sequence analysis. The expression of DAZ proteins in human testis was determined by immunoblot and compared with DAZ cDNA expression. RESULTS: Immunoblot analysis revealed four DAZ protein bands in testis samples that showed no deletions in the AZFc region. No specific bands were observed in samples from AZFc deletion patients. Testis samples from individuals with the partial AZFc deletion, gr/gr, showed two DAZ-specific bands. Interestingly, the sizes of DAZ-specific bands varied among individuals. Analysis of DAZ transcripts in testis samples revealed that the DAZ proteins were translated from the largest of the multiple transcripts originating from each single DAZ gene. CONCLUSIONS: All four DAZ genes are expressed in the human testis, and their products are highly polymorphic among men.
Subject(s)
Infertility, Male/genetics , RNA-Binding Proteins/genetics , Testis/physiology , Antibody Specificity , Cell Line , Chromosomes, Human, Y , Cloning, Molecular , Deleted in Azoospermia 1 Protein , Gene Deletion , Gene Dosage , Humans , Immunohistochemistry , Male , Polymorphism, Genetic , RNA-Binding Proteins/immunology , RNA-Binding Proteins/metabolism , TransfectionABSTRACT
The folding of aptamer immobilized on an Au electrode was successfully detected using label-free electrochemical methods. A thrombin binding DNA aptamer was used as a model system in the presence of various monovalent cations. Impedance spectra showed that the extent to which monovalent cations assist in folding of aptamer is ordered as K(+) > NH(4)(+) > Na(+) > Cs(+). Our XPS analysis also showed that K(+) and NH(4)(+) caused a conformational change of the aptamer in which it forms a stable complex with these monovalent ions. Impedance results for the interaction between aptamer and thrombin indicated that thrombin interacts more with folded aptamer than with unfolded aptamer. The EQCM technique provided a quantitative analysis of these results. In particular, the present impedance results showed that thrombin participates a folding of aptamer to some extent, and XPS analysis confirmed that thrombin stabilizes and induces the folding of aptamer.
