TF2 binds to the regulatory promoter of alkaline phosphatase in Dicytostelium.

Alkaline phosphatase (ALP) activity becomes restricted to PstO cells at the prestalk-prespore boundary during the later stages of development, suggesting a novel function in the regulation of prestalk cell differentiation. To identify regulatory control sequences within the alp promoter, a series of 5' and internal deletions were generated and fused to the LacZ reporter gene. In vitro assays of reporter activity from Dicytostelium transformants containing the deleted promoter-LacZ fusion constructs showed that the -683 to -468 bp sequence is required for proper activation of the reporter in developing slugs. To identify DNA-protein interactions involved in the regulation of alp, EMSAs were preformed using a series of short overlapping PCR probes that span the regulatory promoter sequence. A sequence-specific DNA-binding protein was identified that interacts with the -665 to -635 bp sequence. This DNA-binding protein was sequentially purified using DEAE-Sephacel, heparin-Sepharose, DNA Affinity, and gel filtration chromatography. A polypeptide with a molecular weight of 28 kDa was identified on an SDS-PAGE. The purified protein was identified as TF2 by mass spectrometry. TF2 may, therefore, bind to the regulatory promoter of alp and function in the developmental control of PstO differentiation in Dicytostelium.

Sudden sensorineural hearing loss and antiphospholipid syndrome.

The antiphospholipid syndrome is the association between the presence of antiphospholipid antibodies, thrombosis and/or pregnancy morbidity and mortality. This report presents two cases of antiphospholipid antibodies and sensorineural hearing loss and discusses the probable causative link. We recommend that patients presenting with sudden sensorineural hearing loss are investigated for evidence of antiphospholipid antibodies. Life long anticoagulation is necessary to prevent life threatening thrombotic or thromboembolic events.

Identification and purification of a DNA-binding protein interacting with the promoter of 5'-nucleotidase in Dictyostelium discoideum.

The developmental management of 5'-nucleotidase (5nt) expression in Dictyostelium discoideum has provided a focal point for studies of gene regulation at the level of transcription. To identify DNA-protein interactions involved in the 5nt regulation, EMSAs were performed using short oligonucleotides, designed to span a 357bp promoter region. A binding activity (R(f)=0.33) was identified and shown to be specific to the nucleotide sequence between -338 and -309bp relative to 5nt ATG. Characterization of the binding activity, including the effects of salt and temperature, provided insight into the nature and stability of the protein. The protein was purified in a series of chromatographic stages, including DEAE-Sephacel, heparin-Sepharose, DNA affinity, and gel filtration. SDS-PAGE analysis identified a polypeptide with a molecular weight of 70kDa. Mass spectrometry revealed that the purified protein was a putative formyltetrahydrofolate synthase.