Biomedical subjects

M J Berry

Publications and source records attributed to M J Berry.

At least 55 records · Page 3Linked to original sources

PubMed1997

Noncontact holmium:YAG laser thermal keratoplasty to correct hyperopia: 18-month follow-up.

PURPOSE: To assess the safety and efficacy of noncontact holmium:yttrium aluminium garnet laser thermal keratoplasty (Ho:YAG LTK) for correction of low to moderate hyperopia. METHODS: We performed noncontact Ho:YAG LTK on 1 eye each of 28 patients for correction of hyperopia up to +3.88 dpt. Treatments were conducted with 1 or 2 symmetrical octagonal rings of 8 spots/ring with centerline diameters of 6 mm (1 ring) or 6 and 7 mm (2 rings), 10 pulses of laser light at 5 Hz pulse repetition frequency, variable pulse energy in the range of 208-242 mJ and a nominal spot diameter between 615 and 623 microns. RESULTS: At 18 months after surgery, 20 of 22 (91%) treated patient eyes had improved uncorrected distance visual acuity. The mean change in subjective manifest refraction (spherical equivalent) was -0.52 +/- 0.35 dpt and -1.41 +/- 0.53 dpt for 1- and 2-ring treatment groups, respectively, with good stability in the refractive change after 6 months. The mean induced refractive astigmatism was small (0.30 +/- 0.37 dpt/0.25 +/- 0.29 dpt for 1-/2-ring treatments). None of the eyes lost 2 or more lines of spectacle-corrected distance visual acuity. There were no clinically significant changes in glare and contrast sensitivity. CONCLUSIONS: Noncontact LTK treatment of low hyperopia is safe and effective, and it is more stable and less prone to induce astigmatism than previously reported contact mode LTK treatments.

Adult↗

PubMed1997

Structure-activity relationships for thyroid hormone deiodination by mammalian type I iodothyronine deiodinases.

The bioactivity of thyroid hormone is determined to a large extent by the monodeiodination of the prohormone T4 by the hepatic selenoenzyme type I iodothyronine deiodinase (IDI), i.e. by outer ring deiodination (ORD) to the active hormone T3' or by inner ring deiodination (IRD) to the inactive metabolite rT3. IDI also catalyzes the IRD of T3 and the ORD of rT3' both to T2, as well as the deiodination of different iodothyronine sulfates, e.g. IRD of T3S and ORD of T2S. Previous studies have indicated important differences in catalytic specificity between dog IDI (dID1) and human ID1 (hID1), in particular with respect to the ORD of rT3. This study was done to investigate the relationship between structure and catalytic function of this enzyme by comparing the deiodination of T4, T3, rT3, T3S, and T2S by native dID1 and hID1 in liver microsomes as well as by recombinant wild-type, chimeric and mutated d/hID1 enzymes expressed in HEK293 cells. With both native and recombinant wild-type enzymes, the substrate specificity was T3S > T2S approximately rT3 approximately T4 > T3 for dID1, and rT3 > > T2S approximately T3S > T4 approximately T3 for hID1. Whereas ORD of T4 and of T4, T3, and T3S showed relatively little variation between the different d/hID1 constructs, large differences were found for the ORD of rT3 and T2S. Both reactions were favored by the presence of the amino acids G, E and, in particular, F, present in hID1 at positions 45, 46, and 65, instead of the dID1 residues N, G, and L, respectively. However, although ORD of rT3 was not affected by the presence (hID1) or absence (dID1) of the TGMTR(48-52) sequence, the ORD of T2S was markedly inhibited by the presence of this sequence. Therefore, we have identified structural elements in ID1 that have substrate-specific impacts on deiodination. Our results suggest the specific interaction of the mono-substituted inner ring of the substrates rT3 and T2S but not the disubstituted inner ring of T3, T3S, or T4, with the aromatic ring of F65 in Id1, perhaps by pi-pi interactions.

Amino Acid Sequence↗

PubMed1997

The role of the active site cysteine in catalysis by type 1 iodothyronine deiodinase.

Type 1 iodothyronine deiodinase (deiodinase 1) is a selenoenzyme that converts the prohormone T4 to the active thyroid hormone T3 by outer ring deiodination or to the inactive metabolite rT3 by inner ring deiodination. Although selenocysteine has been demonstrated to be essential for the biochemical profile of deiodinase 1, the role of a highly conserved, active site cysteine (C124 in rat deiodinase 1) has not been defined. The present studies examined the effects of a Cys124Ala mutation on rat deiodinase 1 enzymatic function and substrate affinity. At a constant 10-mM concentration of dithiothreitol (DTT), the C124A mutant demonstrated a 2-fold lower apparent maximal velocity (Vmax) and Km for rT3 (KmrT3) than the wild type for outer ring deiodination, whereas the Vmax/Km ratio was unchanged. Similarly, the apparent Vmax and KmT3 sulfate for inner ring deiodination were 2-fold lower in the C124A mutant relative to those in the wild type, with no change in the Vmax/Km ratio. The C124A mutant exhibited ping-pong kinetics in the presence of DTT, and substitution of the active site cysteine increased the KmDTT by 14-fold relative to that of the wild-type enzyme, with no significant effects on KmrT3 or Vmax. The C124A mutant was inhibited by propylthiouracil in an uncompetitive fashion and exhibited a 2-fold increase in K(i)propylthiouracil compared with that of the wild type. KmrT3 was also reduced for the C124A mutant when 5 mM reduced glutathione, a potential physiological monothiol cosubstrate, was used in outer ring deiodination assays. These results demonstrate that thiol cosubstrate interactions with C124 in type 1 deiodinase play an important role in enhancing catalytic efficiency for both outer and inner ring deiodination.

Amino Acid Sequence↗

PubMed1997

RNA and protein requirements for eukaryotic selenoprotein synthesis.

Selenium has been recognized as an essential nutrient in animals since the 1950s. Demonstration of the role of dietary selenium in protection from oxidative stress followed in the early 1970s, and was largely attributed to its presence as an integral part of cellular glutathione peroxidase. However, the functions of this enzyme did not explain many of the other effects of selenium deficiency. The identification of other mammalian selenoproteins during the last few years has provided new insights into the functions of this trace nutrient. The discovery that type 1 deiodinase (D1) is a selenoenzyme, in addition to unveiling an essential role for selenium in thyroid hormone action, has had more far-reaching implications. Studies of this protein opened the door for investigation of the requirements for eukaryotic selenoprotein synthesis, and the features that distinguish this pathway from the corresponding prokaryotic pathway. Selenium is present in a number of prokaryotic and eukaryotic proteins in the form of the unusual amino acid, selenocysteine. Incorporation of selenocysteine into these proteins requires a novel translation step in which UGA specifies selenocysteine insertion. Since UGA codons are typically recognized as translation stop signals, an intriguing question is raised: How does a cell recognize and distinguish a UGA selenocysteine codon from a UGA stop codon? In this review, we will focus on what is known about selenocysteine incorporation in eukaryotes, briefly summarizing initial studies and discussing a few recent advances in our understanding of this unique "recoding" process.

Animals↗

PubMed1997

Exercise-induced ventilatory abnormalities in orthotopic heart transplant patients.

STUDY OBJECTIVE: The purpose of this investigation was to compare multiple ventilatory responses of heart transplant patients (HTP) with normal subjects (NL) at rest, at absolute and relative submaximal exercise levels, and at peak exercise. DESIGN: Ten male HTP and 10 matched NL were tested under similar conditions on a treadmill with the use of an incremental protocol to symptom-limited maximal levels while breath-by-breath measurements of gas exchange and ventilation were obtained. RESULTS: At an absolute carbon dioxide (VCO2) level of 1 L.min-1, minute ventilation (VE) was significantly higher in HTP compared with NL (38.4 +/- 1.9 versus 29.3 +/- 0.9 L.min-1). However, when compared at similar relative levels (i.e., 40% and 60% of the peak oxygen consumption [VO2 peak]), VE was found to be significantly higher in NL compared with HTP (25.5 +/- 1.4 versus 21.2 +/- 1.0 L.min-1 at 40%; 39.9 +/- 3.1 versus 32.1 +/- 2.0 L.min-1 at 60%, respectively). The reduced VE was the result of a significantly lower tidal volume (VT) in HTP compared with NL at 40% (1.14 +/- 0.08 versus 1.33 +/- 0.05 L) and 60% (1.40 +/- 0.10 versus 1.85 +/- 0.06 L) of VO2 peak, since breathing frequency (BF) was not different between the groups at these levels. CONCLUSIONS: These data demonstrate that HTP have abnormal ventilatory responses to incremental exercise that are largely explained by a diminished VT response. While mechanical factors known to affect VT cannot be ignored, it is likely that the abnormal VT response of HTP during exercise is secondary to respiratory muscle weakness and may be due to hypoperfusion, long-term deconditioning, and/or the long-term use of corticosteriods.

Adult↗

PubMed1996

Human lipoprotein lipase last exon is not translated, in contrast to lower vertebrates.

We have sequenced the first fish (zebrafish, Brachydanio rerio) lipoprotein lipase (LPL) cDNA clone. Similarities were found in mammalian LPL cDNA, but the codon spanning the last two exons (which is thus split by the last intron) is AGA (Arg) as opposed to TGA in mammals. Exon 10 is thus partially translated. These results were confirmed with rainbow trout (Oncorhynchus mykiss). We also investigated whether mammal TGA coded for selenocystein (SeCys), the 21st amino acid, but found that this was not the case: TGA does not encode SeCys but is a stop codon. It thus appears that the sense codon AGA (fish) has been transformed into a stop codon TGA (human) during the course of evolution. It remains to be determined if the "loss" of the C-terminal end of mammalian LPL protein has conferred an advantage in terms of LPL activity or, on the contrary, a disadvantage (e.g., susceptibility to diabetes or atherosclerosis).

Animals↗

PubMed1996

Increased ventilation in runners during running as compared to walking at similar metabolic rates.

At similar levels of carbon dioxide production (VCO2) and oxygen consumption (VO2), runners have been shown to have a greater minute ventilation (VE) during running as compared to walking. The mechanism responsible for these differences has yet to be identified. To determine if these differences are a result of differences in acid-base status, potassium (K+), norepinephrine and/or epinephrine levels, seven well-trained runners completed walk and run tests at similar VO2 and VCO2 levels. The occurrence of entrainment of the breathing and stride frequencies during both walking and running was also determined. VE was significantly greater during the run as compared to the walk, 73.7 (2.2) versus 68.6 (2.0) l.min-1, respectively, despite the similarity in VO2 and VCO2 levels. Alveolar ventilation was not significantly different between the run and the walk, 60.4 (4.7) versus 59.6 (4.4) l.min-1, respectively. Dead space ventilation was found to be significantly greater during running as compared to walking, 13.3 (3.2) versus 9.0 (4.7) l.min-1, respectively. The increases in VE were due to increases in breathing frequency and decreases in tidal volume during the run as compared to the walk. Arterial partial pressures of CO2 (PaCO2) were not significantly different when comparing walking and running to rest values nor when comparing walking and running. Arterial pH was significantly lower during walking as compared to rest and running. Bicarbonate levels were significantly lower during walking as compared to rest. Lactate was significantly greater during walking as compared to rest and to running. K+ levels were significantly higher during walking and running as compared to rest. Epinephrine and norepinephrine levels were not significantly different between running and walking. During the walk, six of the seven subjects entrained their breathing frequency to the stride frequency, and during the run three of the seven subjects demonstrated entrainment. Results from this investigation do not support mediation of VE under the present experimental conditions by changes in arterial levels of humoral factors previously shown to influence VE.

Acid-Base Equilibrium↗

PubMed1996

Estimation of VO2 in older individuals with osteoarthritis of the knee and cardiovascular disease.

The American College of Sports Medicine (ACSM) equation for estimating oxygen consumption (VO2) is often inappropriately applied to non-steady-state treadmill exercise. Therefore, it was the purpose of this investigation to develop an equation to estimate VO2 that could be applied to non-steady-state treadmill exercise in a population of patients with osteoarthritis of the knee, and to assess the generalizability of this equation for estimating VO2peak in patients with cardiovascular disease. Subjects for the investigation were 414 participants in the Fitness and Arthritis in Seniors Trial (FAST), and 362 patients with cardiovascular disease. Results from the FAST subjects showed that the ACSM equation was inappropriate for estimating VO2 during non-steady-state incremental treadmill walking. We developed the following equation (FAST) using speed and the interaction between speed and grade as the predictor variables during treadmill walking: VO2(ml.kg-1.min-1) = 0.0698 x speed(m.min-1) + 0.8147 x grade(%) x speed(m.min-1) + 7.533 ml.kg-1.min-1 The generalizability of the FAST equation for estimating VO2peak was evaluated in the patients with cardiovascular disease. The measured VO2peak of these patients was 23.7 +/- 0.3 ml.kg-1.min-1, whereas the VO2peak values estimated from the FAST equation and the ACSM equation were 24.1 +/- 0.3 and 33.2 +/- 0.5 ml.kg-1.min-1, respectively. No significant differences were found between the measured VO2peak and that estimated from the FAST equation. The VO2peak estimated from the ACSM equation was significantly greater than the measured VO2peak. These results suggest it is more appropriate to use the FAST equation rather than the ACSM equation to estimate VO2 in older patients with either osteoarthritis of the knee or cardiovascular disease.

Age Factors↗

PubMed1996

Inspiratory muscle training and whole-body reconditioning in chronic obstructive pulmonary disease.

To examine the efficacy of targeted inspiratory muscle training (IMT), 25 patients with moderate COPD were randomly assigned to one of three groups. Eight patients received IMT along with general exercise reconditioning, GER+IMT; nine patients received general exercise reconditioning, GER; eight patients received sham breathing exercises, CONTROL. All groups used a spring-loaded inspiratory muscle trainer; however, the GER and CONTROL groups breathed through these devices at only 15% of their maximal inspiratory pressure. The GER+IMT group increased the load on these devices until at 6 wk the load was equal to 80% of their maximal inspiratory pressure. All patients exercised three times per week for a 12-wk period in supervised sessions. Analysis of covariance revealed no significant differences in spirometric measurements, maximal inspiratory pressure, or maximal oxygen consumption among any of the three groups after the intervention (p > 0.05). Twelve-minute walk distance was significantly greater in the GER+IMT and GER groups than in the CONTROL group (p = 0.03). After the intervention, there was a trend (p = 0.08) for treadmill time to be greater for the GER+IMT and GER groups than for the CONTROL group. Dyspnea ratings at different exercise intensities were not found to be significantly different among the three groups after the intervention. These results demonstrate that GER+IMT and GER alone are equally effective in improving exercise performance in patients with COPD. Additionally, the combination of GER and IMT does not appear to provide any clinically significant improvements in exercise performance or perceptions of dyspnea during exercise when compared with GER alone.

Aged↗

PubMed1996

Selenocysteine incorporation in eukaryotes: insights into mechanism and efficiency from sequence, structure, and spacing proximity studies of the type 1 deiodinase SECIS element.

SECIS elements are stem-loop structures located in the 3' untranslated regions (UTRs) of eukaryotic selenoprotein mRNAs that are required for directing cotranslational selenocysteine incorporation at UGA codons. In prokaryotes, stem-loops mediating selenocysteine incorporation are located immediately downstream of the UGA selenocysteine codon, in the coding region. Previous characterization studies of the mammalian SECIS elements of type 1 deiodinase, glutathione peroxidase, and selenoprotein P showed that conserved nucleotides in the loops and unpaired bulges, and base pairing in the stems are required for SECIS function. These initial studies utilized approximately 175-230-nt segments of the 3'UTRs of the selenoprotein mRNAs. Here we define the minimal functional rat type 1 deiodinase SECIS element, a 45-nt segment, the 5' boundary of which corresponds precisely to the 5'-most critical conserved nucleotide identified previously. We also define base pairing requirements in the stem of this element. In view of the presence of SECIS elements in the open reading frames (ORFs) of bacterial selenoproteins, we examine the effects in the type 1 deiodinase of extending the ORF into the SECIS element, and find that this dramatically inhibits SECIS function. Finally, we define a minimal spacing requirement of 51-111 nt between a eukaryotic UGA selenocysteine codon and SECIS element.

Animals↗

PubMed1996

Knowing when not to stop: selenocysteine incorporation in eukaryotes.

The regulation of translation frequently involves protein-RNA interactions. An intriguing example of this is the alternative decoding of UGA, typically a stop codon, as selenocysteine. Two RNA structures, the mRNA selenocysteine insertion sequence (SECIS element) and a unique selenocysteyl-tRNA, are required for this process. In prokaryotes, a single RNA-binding protein, a selenocysteine-specific elongation factor, interacts with both the tRNA and mRNA to confer decoding. Whether eukaryotes use a similar mechanism is currently the subject of intense investigation.

Base Sequence↗

PubMed1996

Hyperopia correction by noncontact holmium:YAG laser thermal keratoplasty. United States phase IIA clinical study with a 1-year follow-up.

PURPOSE: This study was performed to evaluate the safety and effectiveness of noncontact holmium: YAG (Ho:YAG) laser thermal keratoplasty (LTK) for correcting low to moderate hyperopia. METHODS: Twenty-eight patients were treated unilaterally to correct low to moderate hyperopia (up to +3.88 diopters [D] refractive error) using simultaneous noncontact delivery of Ho:YAG laser energy. Treatment parameters included one or two symmetric octagonal rings of eight spots per ring with centerline diameters of 6 mm (1 ring) or 6 and 7 mm (2 rings), ten pulses of laser light at 5-Hz pulse repetition frequency, and variable pulse energy, ranging from 208 to 242 mJ. Follow-up was 1 year in 26 (93%) of the 28 patients. RESULTS: At 1 year postoperatively, uncorrected distance visual acuity was improved in all patients. The mean change in subjective manifest refraction (+/- spherical equivalent [SE]) was -0.55 +/- 0.33 D and -1.64 +/- 0.61 D for one and two-ring treatment groups, respectively, with good stability in the refractive change after approximately 6 months. In the one-ring treatment group (17 eyes), refractive corrections of -0.50 to -1.13 D were achieved in ten eyes (59%), and seven eyes (41%) were unchanged (within +/- 0.25 D) relative to their preoperative measurements. In the two-ring treatment group, all eight eyes (100%) had substantial refractive corrections (range, -0.75 to -2.50 D). Mean induced refractive astigmatism was 0.25 +/- 0.29 D and 0.47 +/- 0.53 D for one- and two-ring treatments, respectively. None of the eyes lost two or more lines of spectacle-corrected distance visual acuity. These was no clinically significant change in endothelial cell density with respect to preoperative values. Glare and contrast sensitivity testing indicate that peripheral corneal opacities produced by LTK do not degrade vision. The amount of refractive change in each group was correlated with the amount of laser pulse energy. CONCLUSIONS: This initial United States clinical study with 1-year follow-up indicates that noncontact LTK treatment of low hyperopia is safe and effective, providing persistent, though modest, refractive corrections in 59% of the one-ring group and larger, persistent, refractive corrections in 100% of the two-ring group.

Adult↗

PubMed1995

Cloning and functional characterization of human selenophosphate synthetase, an essential component of selenoprotein synthesis.

Selenocysteine is co-translationally incorporated into prokaryotic and eukaryotic selenoproteins at in-frame UGA codons. However, the only component of the eukaryotic selenocysteine incorporation machinery identified to date is the selenocysteine-specific tRNA(Sec). In prokaryotes, selenocysteine is synthesized from seryl-tRNA(Sec) and the active selenium donor, selenophosphate. Selenophosphate is synthesized from selenide and ATP by the selD gene product, selenophosphate synthetase, and is required for selenocysteine synthesis and incorporation into bacterial selenoproteins. We have now cloned human selD and shown that transfection of the human selD cDNA into mammalian cells results in increased selenium labeling of a mammalian selenoprotein, type 1 iodothyronine deiodinase. Despite significant differences between the mechanisms of selenoprotein synthesis in prokaryotes and eukaryotes, human selD weakly complements a bacterial selD mutation, partially restoring selenium incorporation into bacterial selenoproteins. Human selenophosphate synthetase has only 32% homology with the bacterial protein, although a highly homologous region that has similarity to a consensus ATP/GTP binding domain has been identified. Point mutations within this region result in decreased incorporation of selenium into type 1 iodothyronine deiodinase in all but one case. Further analysis revealed that reduced selenium labeling was due to altered ATP binding properties of the mutant selenophosphate synthetases.

Adenosine Triphosphate↗

PubMed1995

Translational termination efficiency in mammals is influenced by the base following the stop codon.

The base following stop codons in mammalian genes is strongly biased, suggesting that it might be important for the termination event. This proposal has been tested experimentally both in vivo by using the human type I iodothyronine deiodinase mRNA and the recoding event at the internal UGA codon and in vitro by measuring the ability of each of the 12 possible 4-base stop signals to direct the eukaryotic polypeptide release factor to release a model peptide, formylmethionine, from the ribosome. The internal UGA in the deiodinase mRNA is used as a codon for incorporation of selenocysteine into the protein. Changing the base following this UGA codon affected the ratio of termination to selenocysteine incorporation in vivo at this codon: 1:3 (C or U) and 3:1 (A or G). These UGAN sequences have the same order of efficiency of termination as was found with the in vitro termination assay (4th base: A approximately G >> C approximately U). The efficiency of in vitro termination varied in the same manner over a 70-fold range for the UAAN series and over an 8-fold range for the UGAN and UAGN series. There is a correlation between the strength of the signals and how frequently they occur at natural termination sites. Together these data suggest that the base following the stop codon influences translational termination efficiency as part of a larger termination signal in the expression of mammalian genes.

Animals↗

PubMed1995

Topological analysis of the integral membrane protein, type 1 iodothyronine deiodinase (D1).

Type 1 iodothyronine deiodinase (D1) is a microsomal selenoenzyme which catalyzes deiodination of thyroxine to 3,5,3'-triiodothyronine. Immunoblotting showed that endogenous hepatic, renal, and transiently expressed D1 remains in microsomes after pH 11.5 treatment. In vitro translation studies using pancreatic microsomes identified a single transmembrane domain with a cytosolic carboxyl-terminal catalytic portion. The transmembrane domain is located between conserved basic amino acids at positions 11 and 12 and a group of charged residues at positions 34-39. A transiently expressed D1 protein in which residues 2-25 were deleted was inactive and not integrated into membranes. Activity was not restored by replacing these residues with transmembrane domains from a cytochrome P450 or type 3 deiodinase enzyme despite their incorporation into membranes. Elimination of the positive charges at positions 11 and 12 reduced the amount of transiently expressed protein by 70%, but the enzyme formed was catalytically normal. Similar results were found after conversion of the Lys-27 in the transmembrane domain to Met or Glu. We conclude that the amino terminus of D1 contains uncleaved signal and stop transfer sequence properties. In addition, positively charged residues at positions 11, 12, and 27 are required for optimal formation of the protein but not for catalysis.

Amino Acid Sequence↗

PubMed1995

Nutritional and hormonal regulation of thyroid hormone deiodinases.

Selenocysteine has been identified in the active center of types 1 and 3 iodothyronine deiodinases, two important enzymes regulating the formation and degradation of the active thyroid hormone, 3,5,3'-triiodothyronine (T3). Selenium is thus required for such complex processes as normal growth, brain development, and metamorphosis, all of which are thyroid hormone dependent. Structural and functional analyses of the type 1 deiodinase mRNA allowed identification of the selenocysteine insertion sequence (SECIS) element, a stem-loop structure in the 3' untranslated region of the mRNA. SECIS elements with conserved sequence and structural features are also present in the 3' untranslated regions of the mRNAs encoding selenoprotein P and the glutathione peroxidase family of selenoproteins. These elements are necessary and sufficient for directing selenocysteine incorporation into the deiodinases and the other mammalian selenoproteins.

Amino Acid Sequence↗

PubMed1995

Structural and functional differences in the dio1 gene in mice with inherited type 1 deiodinase deficiency.

The type 1 deiodinase (D1) provides the major portion of the circulating T3 in vertebrates. In C3H and certain other inbred mice, liver and kidney D1 activity is 5- to 10-fold lower than in the common phenotype, C57. The lower D1 levels are paralleled by a decreased normal-sized dio1 mRNA and hyperthyroxinemia. Low activity cosegregates with a restriction fragment length variant (RFLV) in both inbred and recombinant strains, indicating it is due to differences in the dio1 gene. The exonic structure and the deduced amino acid sequences are identical for both strains and highly homologous to that of the rat. The RFLV is due to an approximately 150-base pair expansion of repetitive sequences in the second intron of the C3H gene, but this segment does not differentially affect the transient expression of a human GH gene. The promoter and 5'-flanking regions of the C3H and C57 dio1 genes are very similar and are GC rich without TATA or CCAAT boxes. However, functional assays of 1.5-kilobase 5'-flanking dio1-CAT constructs showed 2- to 3-fold higher activity of the C57-CAT constructs. Deletion mutants showed that sequences between -705 and -162 were the cause of this. In this region, the only major difference between the two genes is a 21-base pair insert containing five CTG repeats in the C3H promoter. This difference also cosegregates with low D1 activity and the intron RFLV in four other mouse strains. The correlation of the CTG repeat insert with both in vitro and in vivo expression and the absence of other significant sequence differences in the 5'-flanking region argue that this is the major explanation for the impaired expression of the dio1 gene and the resulting hyperthyroxinemia of the C3H mouse.

Amino Acid Sequence↗

PubMed1994

Selenocysteine insertion or termination: factors affecting UGA codon fate and complementary anticodon:codon mutations.

Translation of UGA as selenocysteine instead of termination occurs in numerous proteins, and the process of recording UGA requires specific signals in the corresponding mRNAs. In eukaryotes, stem-loops in the 3' untranslated region of the mRNAs confer this function. Despite the presence of these signals, selenocysteine incorporation is inefficient. To investigate the reason for this, we examined the effects of the amount of deiodinase cDNA on UGA readthrough in transfected cells, quantitating the full-length and UGA terminated products by Western blotting. The gene for the selenocysteine-specific tRNA was also cotransfected to determine if it was limiting. We find that the concentrations of both the selenoprotein DNA and the tRNA affect the ratio of selenocysteine incorporation to termination. Selenium depletion was also found to decrease readthrough. The fact that the truncated peptide is synthesized intracellularly demonstrates unequivocally that UGA can serve as both a stop and a selenocysteine codon in a single mRNA. Mutation of UGA to UAA (stop) or UUA (leucine) in the deiodinase mRNA abolishes deiodinase activity; but activity is partially restored when selenocysteine tRNAs containing complementary mutations are contransfected. Thus, UGA is not essential for selenocysteine incorporation in mammalian cells, provided that codon:anticodon complementarity is maintained.

Anticodon↗