Defective RNA splicing resulting from a mutation in the cyclic guanosine monophosphate-phosphodiesterase beta-subunit gene.

PURPOSE: The authors have previously reported a 3' splice site mutation in intron 2 of the rod cyclic guanosine monophosphate-phosphodiesterase (cGMP) beta-subunit (beta-PDE) gene in a patient with compound heterozygous autosomal recessive retinitis pigmentosa. The purpose of this study is to determine whether this mutation interferes with RNA splicing, what products it generates, and whether the resultant mRNA is able to support the synthesis of the native protein. METHODS: Two expression constructs were prepared by subcloning genomic DNA fragments (one from the control subject DNA and the other from the patient's DNA) to the pCIS2 expression vector. Recombinant plasmid DNA was introduced into 293 human embryonic kidney cells using the calcium phosphate-mediated transfection procedure. Northern blot hybridization, reverse transcription-polymerase chain reaction, and sequencing were used for RNA analysis. RESULTS: Four major products were present in the RNA pool isolated from cells transfected with the expression construct containing the splice site mutation. One of the transcripts resulted from the activation of a cryptic 3' splice site located in exon 3, 12 nucleotides downstream of the mutated site. The second fragment was longer than the correctly spliced mRNA by approximately 1 kb and contained unspliced intron 2. Two other high molecular weight products corresponded to intermediate lariats. CONCLUSIONS: An acceptor splice site mutation in intron 2 of the beta-PDE gene leads to the accumulation of pre-mRNA and intermediate lariats and a 12-nucleotide shorter beta-PDE transcript produced by the use of a cryptic splice site located in exon 3. In the normal beta-PDE mRNA, these 12 nucleotides code for ValPheLeuLys. These amino acids are highly conserved in the putative noncatalytic cGMP-binding domain I of beta-PDE from several species and, probably, are important for the correct folding and function of the protein.

Characterization of Key Glycolytic and Oxidative Enzymes in Steinernema carpocapsae.

The enzyme activities of isocitrate dehydrogenase (ICDH, NADP-specific), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), phosphoenolpyruvate carboxykinase (PEPCK), phosphofructokinase (PFK), pyruvate kinase (PK), and fructose-l,6-bisphosphatase (FBPase) were studied in the third-stage juveniles of Steinernema carpocapsae. Reaction requirements, pH optima, substrate and cofactor kinetic constants were similar to those reported previously from other parasitic helminths with the exception of LDH, which was unstable and could not be characterized for specific activity and kinetic constants. The respective pH optima were 7.5 for ICDH, 8.8 for MDH, 6.5 for PEPCK, 7.3 for PFK, 7.2 for PK, and 7.5 for FBPase. The specific activities for ICDH, MDH, PEPCK, PFK, PK, and FBPase at pH 7.5 were 4.8, 1,300, 22, 25, 35, and 6.8 (nmoles substrate min(1) mg protein(1)), respectively. In summary, the infective juveniles of S. carpocapsae display the metabolism typical of a facultative aerobe.

Detection and analysis of human papillomavirus type 1 infection of skin warts from a dermatology clinic in Taiwan by southern hybridization.

Thirty cases of skin warts from the Dermatology Clinic at Tri-Service General Hospital were analyzed for the presence of human papillomavirus type 1 sequences by Southern blot hybridization. Thirteen of the 30 cases were HPV-1 positive. The prevalence was 43%. Episomal HPV-1 sequences were detected in 11 of 13 HPV-1 positive cases. There are two cases which probably contained integrated forms, one contained the higher molecular weight bands, and the other with 6 kb viral genome may be the result of rearrangement and deletion. Histological studies from HPV-1 positive specimens also indicated the typical features of HPV infection. Some cases with high copy number had a high frequency of inclusion bodies. Chi-square analysis showed that HPV-1 prevalence is not related to sex and different lesion locations, but the prevalence of HPV-1 in recurrent lesions is higher than that of initial lesions.

Detection of acute myocardial infarction by evaluation of ultrasonic gray levels in dogs.

The study tested whether an experimental myocardial infarction can be detected from two-dimensional echocardiograms (2DE) by analysis of regional gray levels. The mid-left anterior descending coronary artery was ligated for 3 hours in 14 dogs (group 1) and for 5 hours in 6 dogs (group 2). 2DE were performed before, and after 3 and 5 hours of coronary artery ligation. The ultrasonic amplitude of the control and infarcted regions were obtained from digitized 2DE in the short axis view, at the mid-papillary muscle level. The mean gray levels (+/- SD) of the control and infarcted regions were compared during end-diastolic stop frames. After sacrifice, the hearts were cut into 1 cm thick slices and stained with 1% triphenyl tetrazolium chloride (TTC) solution. The myocardium was then studied by light microscopy. Computerized tomographic scans were also obtained in vitro from 3 hearts of both groups. There was no difference in mean gray levels of the control region during the experiments. However, in the region of wall motion abnormality (area of infarction), the mean gray levels increased from 49.9 +/- 3.5 (before ligation) to 62.0 +/- 7.4 (after 3 hours of ligation, p less than 0.005) in 10 group 1 dogs, but no differences were seen in mean gray levels (49.6 +/- 3.8 vs 50.4 +/- 4.0) in those without a myocardial infarction in 4 group 1 dogs; gray levels also increased from 50.4 +/- 2.9 (before ligation) to 58.6 +/- 6.1 (after 3 hours of ligation, p less than 0.05) and to 65.0 +/- 4.2 (after 5 hours of ligation, p less than 0.005) in group 2 dogs. The area of left ventricular asynergy corresponded precisely to the area of myocardial infarction, determined by both TTC staining and the computerized tomographic scans. The light microscopy of the infarcted area also demonstrated interstitial edema and polymorphonuclear cell infiltration.