Differential translation of Dazap1 transcripts during spermatogenesis.

Deleted in AZoospermia Associated Protein 1 (DAZAP1) is a ubiquitous hnRNP protein that has been implicated in RNA transcription, splicing, and translation. It is highly expressed in testes, predominantly in late stage spermatocytes and post-meiotic spermatids. Dazap1 deficiency in mice results in growth retardation and spermatogenic arrest. The gene produces two major transcripts of 2.4 and 1.8 kb, designated Dazap1-L and Dazap1-S, respectively. Results of our previous RNA in situ hybridization and immunostaining suggested translational regulation of the Dazap1 transcripts during spermatogenesis. The main objectives of the study were to determine the origin of the two Dazap1 transcripts and to investigate whether they were similarly translated. Our Northern and 3' RACE analyses showed that the two transcripts were generated through alternative polyadenylation. In mouse testes, the levels of both transcripts were low at postnatal day 12 (P12), increased significantly at P18, and reached maximum at P27. Sucrose gradient analyses showed that at P12 both transcripts were actively translated. Afterward, an increasing portion of Dazap1-S became associated with the translationally inactive mRNPs, and the translational repression was accompanied by an increase in the length of its poly(A) tail. A much smaller portion of Dazap1-L was also sequestered to mRNPs as testes matured, but there was no changes in its poly(A) tail length. Using RNA pull-down followed by mass spectrometry, we identified DAZL, a germ-cell specific translation regulator, as one of the proteins that bound to the 3'UTR region specific for Dazap1-L. We further showed that DAZL preferentially bound to Dazap1-L in testis lysates and stimulated the translation of a reporter gene carrying Dazap1-L 3'UTR. In summary, our study shows that the translation of the two Dazap1 transcripts is differentially regulated. It also provides a new example of translational repression associated with poly(A) tail elongation during spermatogenesis.

Detection of multiple network-based allosteric interactions between peptides and arrays of DNA binding sites.

Quantitative DNase I footprinting shows that three designed peptides containing N-methylpyrrole (Py) moieties display different types of network-based allosteric communication in binding to DNA: circuit type, incomplete-circuit type, and non-circuit type characterized by interstrand bidentate interactions. Positive cooperative binding of all three peptides to individual DNA binding sites is commonly observed. CD spectral characterization of the interaction between peptides and model undecanucleotide duplexes is consistent with the footprinting results and supports the allosteric model. This study provides insights relating to the interaction network nature of allostery in complex DNA-small molecule interactions.

Expression of recombinant Hirudin in transgenic mice milk driven by the goat beta-casein promoter.

Hirudin, isolated from the leech Hirudo medicinalis, inhibits thrombin directly and several expression systems have been used to produce recombinant Hirudin (rHirudin) for pharmaceutical purposes. A DNA fragment containing the Hirudin coding sequence and goat beta-casein secretion signal was chemically synthesized in this study. The synthetic DNA then was further constructed into a goat beta-casein expression vector for mouse transgenesis. Four lines of transgenic mice were successfully developed and one line showed a meaningful anti-thrombin activity of 40,000 anti-thrombin units (ATU)/mL in their milk. In this animal line, Hirudin mRNA was found in samples of uterus and kidney with insignificant anti-thrombin activity (