Cloning and characterization of hTAFII18, hTAFII20 and hTAFII28: three subunits of the human transcription factor TFIID.

We have cloned cDNAs encoding three novel TAFIIs [TATA-binding protein (TBP)-associated factors] from the human (h) HeLa cell TFIID complexes hTAFII28, hTAFII20 and hTAFII18. hTAFII28 is a core hTAFII present in both of the previously described hTFIID species which either lack or contain hTAFII30 (hTFIID alpha and hTFIID beta respectively), and is the homologue of Drosophila (d)TAFII30 beta. hTAFII18 is a novel hTAFII which shows homology to the N-terminal region of the yeast TAFIISPT3, but has no known Drosophila counterpart. In contrast to hTAFII28, hTAFII18 is a TFIID beta-specific hTAFII. hTAFII20 is the homologue of p22, an alternatively spliced form of dTAFII30 alpha (p32). Using a combination of protein affinity chromatography and cotransfection and immunoprecipitation assays, we have identified a series of in vitro and intracellular interactions among the novel hTAFIIs and between the novel hTAFIIs and hTAFII30 or TBP. We show that hTAFII28 interacts with hTAFII18 both in vitro and intracellularly; in contrast to its Drosophila homologue, hTAFII28 also interacts directly with TBP. Deletion analysis indicates that TBP and hTAFII18 bind to distinct domains of hTAFII28. hTAFII18 also interacts with TBP, but it interacts more strongly with hTAFII28 and hTAFII30. The binding of hTAFII28 and hTAFII30 requires distinct domains of hTAFII18. As observed with the homologous Drosophila proteins, hTAFII20 interacts directly with TBP; however, additional interactions between hTAFII20 and hTAFII28 or hTAFII30 were detected. These results reveal differences not only in subunit composition, but also in the organization of dTFIID and hTFIID complexes.     

Micropuncture pressure in small arteries or veins of perfused rabbit lungs

Until now, direct micropuncture measurements of vascular pressure in lung have been limited to small vessels less than 100 microns on the pleural surface. On the other hand, direct pressure measurements using small catheters (less than 1-mm OD) in pulmonary vessels have been limited to those greater than 1.2 mm. We measured pressure in intermediate-sized microvessels (300-700 microns) using the micropuncture method in isolated perfused rabbit lungs. These microvessels are located 2 or 3 mm beneath the pleura. We exposed them by microsurgery and punctured the relatively thick-walled vessels with specially configured micropipettes. We exposed one pulmonary microvessel in each rabbit lung by microsurgery on the left middle lobe. In 15 rabbit lungs we measured pressure in a total of six small arteries (275- to 470-microns diam) and nine small veins (300- to 700-microns diam) under high zone 3 conditions, near the zone 2/3 boundary. We found approximately 35% of the total pulmonary vascular pressure drop in arteries greater than 275-microns diam and 7% in veins greater than 300-microns diam. In veins greater than 500-microns diam, there was no measurable pressure drop. After the measurements, we froze the lung and confirmed that there was no detectable interstitial or alveolar edema in the cross sections of the punctured site. Our data are compatible with those of other investigators who have used isolated perfused rabbit lungs under similar experimental conditions.