Expression in bacteria of functional inhibitory subunit of retinal rod cGMP phosphodiesterase.

The cGMP phosphodiesterase of vertebrate retinal rod outer segments plays a key role in visual transduction. A functionally active form of the inhibitory gamma subunit of the phosphodiesterase, which keeps the enzyme inactive in the dark, has been obtained in high yield from a synthetic gene expressed in Escherichia coli. A DNA sequence encoding the 87-residue bovine gamma subunit was chemically synthesized and assembled from 10 oligonucleotides. The synthetic gene was cloned into an expression vector that uses the promoter PL of lambda phage. E. coli was transformed with this vector, which encodes a fusion protein consisting of the first 31 residues of the lambda cII protein, a 7-residue joining sequence that is specifically cleaved at its C-terminal end by clotting protease factor Xa, and the 87-residue gamma subunit. The fusion protein was solubilized in 6 M urea and purified by ion-exchange chromatography on a CM-Sephadex column. The typical yield was 1 mg of fusion protein per liter of bacterial culture, which corresponds to the amount of gamma in about 2500 bovine retinas. Proteolytic cleavage of the fusion protein by factor Xa released a synthetic gamma with the same amino acid sequence as that of native gamma. Both fusion protein and synthetic gamma inhibited trypsin-activated phosphodiesterase with high affinity (Kd less than 100 pM). Likewise, both were as effective as native gamma in inhibiting transducin-activated phosphodiesterase in rod outer segment membranes. This inhibition was reversed by the activation of additional transducin. Thus, the N terminus of gamma is not intimately involved in interactions with either the catalytic subunits of the phosphodiesterase or the activated form of transducin. In contrast, a C-terminal deletion mutant terminating at residue 74 of gamma stimulated rather than inhibited the trypsin-activated enzyme. Thus, the C-terminal region of gamma is critical for inhibition of the phosphodiesterase.     

cGMP phosphodiesterase of retinal rods is regulated by two inhibitory subunits.

The cGMP phosphodiesterase (PDE) of cattle retinal rod outer segments comprises three types of subunits: the two heavy catalytic ones, PDE alpha and PDE beta, each around 85 kDa, and the light inhibitory one, PDE gamma or I (11 kDa). The relative stoichiometry is usually assumed to be 1:1:1. PDE activation in the visual transduction cascade results from removal of the inhibitor by the alpha subunit of transducin (T alpha). The stoichiometric complex T alpha-I, separated from activated PDE, has been isolated and characterized. Analyzing now the activated PDE, we find that it still contains some inhibitor and is resolvable into two species, one with 50% of the inhibitor content of the native enzyme and the other totally devoid of it. The same two species are observed upon activation of PDE by very short tryptic proteolysis, which specifically degrades the inhibitor. This leads us to conclude that the composition of the native enzyme is PDE alpha beta-I2. The two inhibitory subunits are differentially bound, sequentially removable, and exchangeable between the native complex PDE alpha beta-I2 and the fully active PDE alpha beta. The possibility of this exchange precludes as yet an unambiguous estimate of the actual activity of the intermediate complex PDE alpha beta-I. The differential binding and the exchangeability of the inhibitors raises the possibility of a fast, diffusion controlled, switch-off mechanism of PDE activity after a flash, which would shortcut the inactivation resulting from the slow GTPase rate of transducin.     

Retinal waves in mice lacking the beta2 subunit of the nicotinic acetylcholine receptor

The structural and functional properties of the visual system are disrupted in mutant animals lacking the β2 subunit of the nicotinic acetylcholine receptor. In particular, eye-specific retinogeniculate projections do not develop normally in these mutants. It is widely thought that the developing retinas of β2^−/− mutants do not manifest correlated activity, leading to the notion that retinal waves play an instructional role in the formation of eye-specific retinogeniculate projections. By multielectrode array recordings, we show here that the β2^−/− mutants have robust retinal waves during the formation of eye-specific projections. Unlike in WT animals, however, the mutant retinal waves are propagated by gap junctions rather than cholinergic circuitry. These results indicate that lack of retinal waves cannot account for the abnormalities that have been documented in the retinogeniculate pathway of the β2^−/− mutants and suggest that other factors must contribute to the deficits in the visual system that have been noted in these animals.     

Phosphatidylinositol-stimulated phosphorylation of an inhibitory subunit of cGMP phosphodiesterase in vertebrate rod photoreceptors.

An inhibitory subunit (P gamma) of cGMP phosphodiesterase from vertebrate rod photoreceptors (frog, toad, and bovine) was phosphorylated by cytosolic protein kinase(s) derived from intact frog rod outer segments. The phosphorylation of frog P gamma was stimulated by phosphatidylinositol but not by cAMP or cGMP. One- and two-dimensional gel electrophoresis revealed that 70-80% of P gamma was phosphorylated with 1 mol of phosphate per frog P gamma under optimal conditions. A peptide that derived from an active domain of bovine P gamma was also phosphorylated. Phosphorylation of frog P gamma was inhibited by addition of the peptide to the reaction mixture. Phosphorylation of frog P gamma was also inhibited by addition of transducin subunits or active (P gamma-less) cGMP phosphodiesterase. Okadaic acid, on the other hand, enhanced P gamma phosphorylation, suggesting the presence of protein phosphatase(s) in the cytosolic fraction. These data suggest another mechanism for the regulation of cGMP phosphodiesterase in vertebrate rod photoreceptors.     

Photoreceptor degeneration induced by the expression of simian virus 40 large tumor antigen in the retina of transgenic mice.

Expression of the viral oncogene encoding the simian virus 40 (SV40) large tumor antigen (T antigen) typically promotes tumorigenesis in mammalian cells. To generate transgenic mice that express T antigen in rod photoreceptors, a chimeric construct consisting of a mouse opsin promoter fragment fused to the coding region of SV40 T antigen was generated. Expression of T antigen in the transgenic retina began at early stages of postnatal development concomitant with expression of endogenous opsin. Instead of inducing hyperplasia or tumor formation, T-antigen expression caused a rapidly progressing photoreceptor degeneration. The degeneration was accompanied by sustained DNA synthesis in photoreceptor cells, as evidenced by incorporation of [3H]thymidine and by the appearance of mitotic figures at postnatal day 10, a stage when nontransgenic photoreceptor cells are postmitotic and quiescent. Although transgenic photoreceptor cells undergo S phase and enter mitosis, the consequences of T-antigen expression are not proliferation and tumorigenesis but proliferation and cell death.     

Mechanism of photoreceptor cGMP phosphodiesterase inhibition by its gamma-subunits.

cGMP phosphodiesterase (PDE) is the key effector enzyme of vertebrate photoreceptor cells that regulates the level of the second messenger, cGMP. PDE consists of catalytic alpha and beta subunits (Palpha and Pbeta) and two inhibitory gamma subunits (Pgamma) that block PDE activity in the dark. The major inhibitory region has been localized to the C terminus of Pgamma. The last C-terminal residues -IleIle form an important hydrophobic domain critical for the inhibition of PDE activity. In this study, mutants of Pgamma were designed for cross-linking experiments to identify regions on Palpha and Pbeta subunits that bind to the Pgamma C terminus. In one of the mutants, the cysteine at position 68 was substituted with serine, and the last four C-terminal residues of Pgamma were replaced with a single cysteine. This mutant, Pgamma83Cys, was labeled with photoprobe 4-(N-maleimido) benzophenone (MBP) at the cysteine residue. The labeled Pgamma83CysMBP mutant was a more potent inhibitor of PDE activity than the unlabeled mutant, indicating that the hydrophobic MBP probe mimics the Pgamma hydrophobic C terminus. A specific, high-yield cross-linking of up to 70% was achieved between the Pgamma83CysMBP and PDE catalytic subunits. Palpha and the N-terminally truncated Pbeta (lacking 147 aa residues) cross-linked to Pgamma83CysMBP with the same efficiency. Using mass spectrometric analysis of tryptic fragments from the cross-linked PDE, we identified the site of cross-linking to aa residues 751-763 of Palpha. The corresponding region of Pbeta, Pbeta-749-761, also may bind to the Pgamma C terminus. Our data suggest that Pgamma blocks PDE activity through the binding to the catalytic site of PDE, near the NKXD motif, a consensus sequence for interaction with the guanine ring of cGMP.     

Direct activation of cGMP-dependent channels of retinal rods by the cGMP phosphodiesterase.

The cationic conductances of purified bovine retinal rod membranes were studied by incorporation of vesicles into planar lipid bilayers. When the membranes were stripped of all peripheral proteins [guanine nucleotide-binding protein (G protein) and cGMP phosphodiesterase (3',5'-cyclic-GMP 5'-nucleotidohydrolase), EC 3.1.4.35], sodium and calcium fluxes were almost only observed in the presence of cGMP. Reconstitution experiments in which purified cGMP phosphodiesterase alone or with G protein were reassociated to the vesicles in proportions similar to those found in the native rod provide evidence for a direct interaction between the cGMP-dependent channel protein and the phosphodiesterase. (i) In its inhibited state, phosphodiesterase markedly stimulates the activity of the channels in the presence of cGMP (situation in the dark-adapted rod) but is not capable of activating the channels in the absence of cGMP. (ii) In the absence of cGMP, activation of the phosphodiesterase by G protein with GTP bound (equivalent to photoexcitation) induces the opening of cation channels that have the same conductance for sodium ions as cGMP-activated channels (20-22 pS, with two sublevels of about 7 pS and 13 pS).     

Human beta-globin gene expression in transgenic mice is enhanced by a distant DNase I hypersensitive site.

Several lines of evidence suggest that erythroid-specific DNase I hypersensitive sites (HS) located far upstream of the human beta-globin gene are important in regulating beta-globin gene expression. We used the polymerase chain reaction technique to amplify and clone an 882-base-pair DNA fragment spanning one of these HS, designated HSII, which is located 54 kilobases upstream of the beta-globin gene. The cloned HSII fragment was linked to a human beta-globin gene in either the genomic (HSII-beta) or antigenomic (HSII-beta) orientation. These two constructs and a beta-globin gene alone (beta) were injected into fertilized mouse eggs, and expression was analyzed in liver and brain from day-16 transgenic fetuses. Five of 7 beta-transgenic fetuses expressed human beta-globin mRNA, but the level of expression per gene copy was low, ranging from 0.93 to 22.4% of mouse alpha-globin mRNA (average 9.9%). In contrast, 11 of 12 HSII-beta transgenic fetuses expressed beta-globin mRNA at levels per gene copy ranging from 31.3 to 336.6% of mouse alpha-globin mRNA (average 139.5%). Only three fetuses containing intact copies of the HSII-beta construct were produced. Two of three expressed human beta-globin mRNA at levels per gene copy of 179.2 and 387.1%. Expression of human beta-globin mRNA was tissue-specific in all three types of transgenic fetuses. These studies demonstrate that a small DNA fragment containing a single erythroid-specific HS can stimulate high-level human beta-globin gene expression in transgenic mice.     

Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions.

Aberrant expression of transforming growth factor beta 1 (TGF-beta 1) has been implicated in a number of disease processes, particularly those involving fibrotic and inflammatory lesions. To determine the in vivo effects of overexpression of TGF-beta 1 on the function and structure of hepatic as well as extrahepatic tissues, transgenic mice were generated containing a fusion gene (Alb/TGF-beta 1) consisting of modified porcine TGF-beta 1 cDNA under the control of the regulatory elements of the mouse albumin gene. Five transgenic lines were developed, all of which expressed the Alb/TGF-beta 1 transgene selectively in hepatocytes. The transgenic line 25 expressing the highest level of the transgene in the liver also had high (> 10-fold over control) plasma levels of TGF-beta 1. Hepatic fibrosis and apoptotic death of hepatocytes developed in all the transgenic lines but was more pronounced in line 25. The fibrotic process was characterized by deposition of collagen around individual hepatocytes and within the space of Disse in a radiating linear pattern. Several extrahepatic lesions developed in line 25, including glomerulonephritis and renal failure, arteritis and myocarditis, as well as atrophic changes in pancreas and testis. The results from this transgenic model strongly support the proposed etiological role for TGF-beta 1 in a variety of fibrotic and inflammatory disorders. The transgenic model may also provide an appropriate paradigm for testing therapeutic interventions aimed at neutralizing the detrimental effects of this important cytokine.     

The membrane binding domain of rod cGMP phosphodiesterase is posttranslationally modified by methyl esterification at a C-terminal cysteine.

Retinal rod cGMP phosphodiesterase (3',5'-cyclic-GMP phosphodiesterase; EC 3.1.4.35; PDE), a key regulatory enzyme involved in visual excitation, is one of several outer segment membrane proteins that are carboxyl methylated in the presence of the methyl donor S-adenosyl-L-[3H-methyl]methionine. By chromatographic analyses of the 3H-methyl amino acid generated by exhaustive proteolysis of purified PDE, followed by performic acid oxidation of the digest, we have shown that this modification occurs at a C-terminal cysteine residue of the alpha subunit of this enzyme. When PDE is subjected to limited proteolysis with trypsin, a 3H-methylated fragment of 1000 daltons or less is rapidly removed prior to the degradation of its inhibitory gamma subunit. This small fragment remains membrane bound, whereas the bulk of the enzyme is released, indicating that a domain responsible for anchoring PDE to the membrane is located near the C terminus. Based on the C-terminal amino acid sequence of Cys-Cys-Val-Gln predicted from the alpha cDNA sequence, we conclude that PDE undergoes posttranslational modifications, including the proteolytic removal of two or three terminal amino acids, and methyl esterification of the alpha-carboxyl group of the terminal cysteine residue. We speculate that the sulfhydryl group of the methylated cysteine is also lipidated to mediate membrane binding. These modifications may play an important role in delivering the nascent PDE chains to the membrane and in correctly positioning the PDE molecule in the rod disks for phototransduction.     

High expression of human beta S- and alpha-globins in transgenic mice: hemoglobin composition and hematological consequences.

A line of transgenic mice (alpha H beta S-11; where alpha H is human alpha-globin) was created in which the human beta S and human alpha 2 globin genes, each linked to the beta-globin locus control region, were cointegrated into the mouse genome. On a normal genetic background, the transgenic mice produced 36% human beta S-globin chains with an alpha H/beta S ratio of 1.3. Higher levels of beta S were achieved by breeding the transgenic mice with mutant mice carrying a mouse beta major-globin gene deletion. Mice heterozygous for the beta major deletion (alpha H beta S[beta MD]; MD, mouse deletion) had 54% beta S with an alpha H/beta S ratio of 1.0; mice homozygous for the beta major deletion (alpha H beta S[beta MDD]) had 72.5% beta S and an alpha H/beta S ratio of 0.73. Because mouse alpha chains inhibit hemoglobin (Hb) S polymerization, we bred the mice to heterozygosity for a mouse alpha-globin deletion. These mice (alpha H beta S[alpha MD beta MDD]) had an increased alpha H/beta S ratio of 0.89 but expressed 65% beta S. Expression of the human genes cured the thalassemic phenotype associated with the murine beta major deletion. Transgenic alpha H beta S[beta MDD] mice had normal hematocrit and Hb and somewhat elevated reticulocytes (6% vs. 3% for control), whereas the mice carrying the alpha-globin deletion (alpha H beta S[alpha MD beta MDD]) had a normal hematocrit and Hb and more elevated reticulocytes (10.3 +/- 7.6% vs. 3.4 +/- 1.0%). Expression of the transgene restored a normal distribution of erythrocyte densities when compared to thalassemic mice; however, the average mean corpuscular Hb concentration of alpha H beta S[beta MDD] mice increased to 35.7 g/dl (vs. control 33.7 g/dl) whereas that of alpha H beta S[alpha MD beta MDD] mice was further elevated to 36.3 g/dl. The intrinsic oxygen affinity was increased in transgenic mouse erythrocytes at 280 milliosmolal, and the PO2 at midsaturation of alpha H beta S[alpha MD beta MDD] erythrocytes was higher than that of alpha H beta S[beta MDD] cells (37.4 +/- 2 vs. 33.5 +/- 1 mmHg). The higher values of the mean corpuscular Hb concentration and intrinsic PO2 at midsaturation, which favor in vivo sickling, may explain the slightly more severe hematological picture in alpha H beta S[alpha MD beta MDD] mice. We conclude that the transgenic mouse with high Hb S expression does not exhibit adult anemia but does have abnormal hematological features: increased erythrocyte density, high oxygen affinity, and reticulocytosis with increased stress reticulocytes.     

Pancreatic expression of human insulin gene in transgenic mice.

We have investigated the possibility of obtaining integration and expression of a native human gene in transgenic mice. An 11-kilobase (kb) human chromosomal DNA fragment including the insulin gene (1430 base pairs) was microinjected into fertilized mouse eggs. This fragment was present in the genomic DNA of several developing animals. One transgenic mouse and its progeny were analyzed for expression of the foreign gene. Synthesis and release of human insulin was revealed by detection of the human C-peptide in the plasma and urine. Human insulin mRNA was found in pancreas but not in other tissues. These findings indicate that the 11-kb human DNA fragment carries the sequences necessary for tissue-specific expression of the insulin gene and the human regulatory sequences react to homologous signals in the mouse.     

Suppression of eGFP expression in erythroid-specific transgenic mice by siRNA

We have studied the role of small interfering RNA (siRNA) in the suppression of the enhanced green fluorescent protein (eGFP) in vitro and in vivo. siRNA plasmids for suppressing the eGFP expression were constructed, in which H1 promoter amplified from human 293 T cells was used to drive the small interfering RNA (19-nt) synthesis. In vitro studies demonstrated that the constructed siRNA plasmids had an effective suppressive effect on eGFP expression in 293 T and Mel cells. When the siRNA plasmid was injected into erythroid-specific eGFP transgenic mice, the eGFP expression were significantly suppressed (over 20% reduction) in the recipient mice compared to the control mice and the suppressing effect lasted for 2 weeks after one single injection. Moreover, the suppressive effect could be re-generated or boosted with secondary injections. Our results demonstrate a tissue-specific gene suppression by siRNA treatment.     

Overexpression of the acid-labile subunit of the IGF ternary complex in transgenic mice

The ternary complex, composed of IGF-I or IGF-II, IGF-binding protein-3, and the acid-labile subunit, is responsible for transport of the majority of the IGF-I and IGF-II present in the circulation. Acid-labile subunit is developmentally and hormonally regulated, suggesting an important, although unclear, role in regulating the availability and action of the IGFs. To investigate the biological role of acid-labile subunit, we generated transgenic mice, which constitutively overexpress a human acid-labile subunit cDNA driven by the cytomegalovirus promoter. Two independent transgenic strains, CMVALS-1 and CMVALS-2, with mean serum levels of human acid-labile subunit of 19.3 +/- 4.2 and 20.2 +/- 3.2 microg/ml respectively, were characterized. Total acid-labile subunit, endogenous plus transgene derived, was measured by Western blotting and was found to be significantly increased in transgenic compared with wild-type mice (1.51 +/- 0.02-fold; P < 0.001). There were no significant differences in serum IGF-binding protein-3 or IGF-I levels between transgenic and wild-type mice. Similar chromatographic elution patterns were observed when sera from transgenic and wild-type mice were preincubated with [(125)I]IGF-I, indicating that acid-labile subunit overexpression had no measurable effect on compartmentalization of IGF-I in the circulation. Transgene-derived human acid-labile subunit mRNA was detected in 17-d-old embryos and all adult mouse tissues examined. A significant reduction in litter size was also observed in each of the acid-labile subunit transgenic mouse strains. This reduction in litter size was due to a maternal effect, as it was apparent when transgenic female mice were crossed with wild-type male mice, but not when male transgenic mice were crossed with female wild-type mice. The transgenic mice were phenotypically normal at birth, but demonstrated a significant reduction in postnatal body weight gain, particularly during the first 3 wk of life. Over the first 3 months of life, average body weights were significantly reduced by 5.3 +/- 0.6%, 4.2 +/- 0.6%, 8.1 +/- 0.9%, and 5.6 +/- 0.8%, compared with those in wild-type mice, for male and female CMVALS-1 mice and male and female CMVALS-2 mice, respectively. Double transgenic mice, generated by crossing acid-labile subunit transgenic mice with transgenic mice that overexpress IGF-binding protein-3, demonstrated a significantly more marked reduction in body weight gain than acid-labile subunit transgenic mice. These data demonstrate that overexpression of acid-labile subunit has significant effects on postnatal growth and reproduction. As there is little measurable alteration in the circulating components of the IGF system, these effects are most likely to be mediated via disturbances in tissue IGF availability.