Complete cDNA sequences of mouse rod photoreceptor cGMP phosphodiesterase alpha- and beta-subunits, and identification of beta'-, a putative beta-subunit isozyme produced by alternative splicing of the beta-subunit gene.

We have characterized overlapping cDNA clones encoding cGMP phosphodiesterase (PDE) alpha- and beta-subunits of mouse retinal rod photoreceptors. The open reading frames predict an alpha-subunit of 100 kDa (856 residues), and a beta-subunit of 99 kDa (853 residues). Sequence analysis of two of twelve beta-subunit clones predicts the presence in the retina of an additional PDE, termed beta', which is generated by alternative splicing of the beta-subunit gene. beta' differs from beta only at the C-terminus being 55 residues shorter and lacking the Caax motif found at the C-termini of both the alpha- and beta-subunits. A 300 residue segment thought to contain the active site is present in the C-terminal half of alpha, beta and beta'.

Comparison of H5, S6, and H5-S6 exchanges on pore properties of voltage-dependent K+ channels.

Evidence is now substantial that membrane segments besides H5 contribute to the pore of K+ channels. We found that substitution of the H5 region of Shaker B (ShB) with the corresponding sequence of NGK2 expressed channels which retained the single channel K+ conductance (gK+) of the host ShB channel. A reverse chimera with ShB H5 region transplanted into NGK2 also retained the gK+ of the host NGK2. Point mutations V443L+T449Y in ShB H5 converted internal tetraethylammonium (TEA) affinity to NGK2 values, and T449Y converted external TEA affinity and Rb+ conductance (gRb+) to NGK2 values. In ShB, exchanging a short stretch of 9 amino acids located just past the transmembrane segment referred to as S6, post-S6+ produced a large increase in gK+ with no effect on internal or external TEA blockade. Within S6, 3 important residues for internal TEA blockade were identified. Thus, H5 determines external TEA blockade and both H5 and S6 may determine internal TEA blockade, but neither H5 nor S6 alone restored the donor gK+. However, chimeric channels in which H5, S6, and post-S6 were exchanged transferred gK+ of NGK2 to ShB or the gK+ of ShB to NGK2. Thus, contributions from H5, S6, and its cytoplasmic extension post-S6 make the pore of voltage-dependent Shaker K+ channels a polysegmental mosaic structure.

A comparison of pulmonary arterial occlusion algorithms for estimation of pulmonary capillary pressure.

Using the arterial occlusion method, we compared five literature-based estimates of pulmonary capillary pressure (Ppc) with the corresponding double occlusion pressures (Pdo) in anesthetized dogs whose chests had been closed after sternotomy for instrumentation. Arterial occlusions were performed with a balloon-tipped pulmonary artery catheter that housed pressure transducers immediately proximal and distal to the balloon. Separation of the proximal and distal pressure waveforms during balloon inflation allowed us to precisely define the moment of occlusion. We fit a monoexponential curve to pressure data beginning 200 ms after the onset of occlusion and a biexponential curve to data beginning at the instant of occlusion, with data obtained over a range of vascular states (control, serotonin infusion, histamine infusion). In addition, we investigated the use of sampling of the raw data to estimate capillary pressure. Three of the five literature-based estimates of Ppc yielded values similar to Pdo. The optimal (least average difference from Pdo) interpolation/extrapolation time points of the curve fits varied, depending on the type of curve fitting and the state of the pulmonary vasculature. We also determined that a close approximation of Pdo may be derived from the raw data, as an alternative to exponential curve fitting.

Mouse opsin. Gene structure and molecular basis of multiple transcripts.

The single copy mouse opsin gene produces five major transcripts, varying in size from 1.7 to 5.1 kilobases. The mRNAs are present at levels that vary over 2 orders of magnitude and can be detected as early as postnatal day 1. Each of the transcripts is polyadenylated and can be identified in polysome-bound RNA, suggesting that each is translated in vivo. To elucidate the molecular basis of this complex transcription pattern, we have characterized genomic fragments covering the entire mouse opsin gene, including several kilobases of 5'- and 3'-flanking regions. Transcription initiates at a single site 97 base pairs upstream of the translation start codon. Northern hybridization with exon- and intron-specific probes demonstrated that the various transcripts are not generated by partial or alternative splicing. Sequence analysis of the 3' end of the gene showed the presence of multiple polyadenylation signals. Analysis by polymerase chain reaction of the 3' end of opsin cDNA demonstrated that the complex transcription pattern originated from the selective use of these polyadenylation sites, generating transcripts that differ only in the length of the 3'-untranslated region. Transcript heterogeneity similar to that observed in mouse was also found in rat and, to a lesser degree, in human and frog opsin mRNAs.

Molecular characterization of human and bovine rod photoreceptor cGMP phosphodiesterase alpha-subunit and chromosomal localization of the human gene.

Defects in proteins that function in photoreceptor signal transduction are prime suspects as causes of some human hereditary retinal degenerations. We have characterized cDNA clones encoding the alpha-subunit of human and bovine rod cell cGMP phosphodiesterase a key phototransduction enzyme. Clones from both species contain an open reading frame capable of coding for an approximately 100-kDa polypeptide of 859 amino acids, 94% of which are identical. Two or more transcripts were detected in both human and bovine retinal poly(A)+ RNA preparations, although the human transcripts ranging from 5.3 to 4.9 kb are significantly larger than the two bovine transcripts of 4.6 and 4.0 kb. The bovine and human genes appear to exist in single copy, with the bovine gene spanning more than 140 kb of genomic DNA. Somatic cell hybrids were used to map the human gene to the long arm of chromosome 5 (5q31.2----q34). Finally, the use of the candidate gene approach in the study of hereditary retinal dystrophies is discussed.