Adenine Guanine Research Articles

The RNA splicing factor hSlu7 is required for correct 3' splice-site choice.

The production of correctly spliced messenger RNA requires two catalytic splicing steps. During step II, exon 1 attacks an adenine-guanine (AG) dinucleotide at the 3' splice site. This AG is usually located between 18 and 40 nucleotides downstream from the branch site, and closer AGs are skipped in favour of AGs located more optimally downstream. At present, little is understood about how the correct AG is distinguished from other AGs. Here we describe a metazoan splicing factor (hSlu7) that is required for selection of the correct AG. In the absence of hSlu7, use of the correct AG is suppressed and incorrect AGs are activated. We investigated this loss of fidelity by analysing spliceosomes assembled in the absence of hSlu7. These studies reveal that exon 1 is loosely associated with these spliceosomes. Thus, the improperly held exon cannot access the correct AG, but can attack other AGs indiscriminately. We conclude that hSlu7 is required to hold exon 1 tightly within the spliceosome for attack on a prespecified AG.

Exercise intolerance due to mutations in the cytochrome b gene of mitochondrial DNA.

The mitochondrial myopathies typically affect many organ systems and are associated with mutations in mitochondrial DNA (mtDNA) that are maternally inherited. However, there is also a sporadic form of mitochondrial myopathy in which exercise intolerance is the predominant symptom. We studied the biochemical and molecular characteristics of this sporadic myopathy. We sequenced the mtDNA cytochrome b gene in blood and muscle specimens from five patients with severe exercise intolerance, lactic acidosis in the resting state (in four patients), and biochemical evidence of complex III deficiency. We compared the clinical and molecular features of these patients with those previously described in four other patients with mutations in the cytochrome b gene. We found a total of three different nonsense mutations (G15084A, G15168A, and G15723A), one missense mutation (G14846A), and a 24-bp deletion (from nucleotide 15498 to 15521) in the cytochrome b gene in the five patients. Each of these mutations impairs the enzymatic function of the cytochrome b protein. In these patients and those previously described, the clinical manifestations included progressive exercise intolerance, proximal limb weakness, and in some cases, attacks of myoglobinuria. There was no maternal inheritance and there were no mutations in tissues other than muscle. The absence of these findings suggests that the disorder is due to somatic mutations in myogenic stem cells after germ-layer differentiation. All the point mutations involved the substitution of adenine for guanine, but all were in different locations. The sporadic form of mitochondrial myopathy is associated with somatic mutations in the cytochrome b gene of mtDNA. This myopathy is one cause of the common and often elusive syndrome of exercise intolerance.

Prevalence of macular pattern dystrophy in maternally inherited diabetes and deafness

Objective To evaluate the prevalence of macular pattern dystrophy (MPD) in maternally inherited diabetes and deafness (MIDD), a new subtype of diabetes mellitus that cosegregates with a mutation of mitochondrial DNA (i.e., the substitution of guanine for adenine at position 3243 of leucine transfer RNA) and to report the clinical characteristics of MPD. Design Prospective cohort study. Participants Forty-six patients from 29 families with an adenine-to-guanine mutation of mitochondrial DNA were recruited from a French collaborative multicenter study. Thirty-five patients had MIDD, 8 were asymptomatic children of MIDD patients, and 3 had MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes). The 33 MIDD patients with diabetes were matched for diabetes duration and gender with 33 patients with “common” type-2 diabetes to compare the prevalence of diabetic retinopathy (DR) in both series. Methods All patients had a full ophthalmologic examination and fundus photographs. Main outcome measures The presence and severity of MPD and DR were assessed in each patient. Results Thirty MIDD patients (85.7%) of 35 exhibited bilateral MPD characterized by linear pigmentation surrounding the macula and optic disc. In 24 of these 30 patients, visual acuity was 20/25 or more in both eyes. The prevalence of DR was 6% in MIDD patients with diabetes versus 15% for patients with common type-2 diabetes (a difference that was not significant, P = 0.23). The fundus of each of the eight asymptomatic children was normal. MPD was present in one of the three cases of MELAS. Conclusion The prevalence of MPD in MIDD is high. Its detection may be helpful for the diagnosis of this new subtype of diabetes, for which specific treatments may be proposed.

Renal complications in patients with diabetes mellitus associated with an A to G mutation of mitochondrial DNA at the 3243 position of leucine tRNA

The substitution of guanine for adenine at position 3243 of the leucine tRNA gene of mitochondrial DNA was originally described in association with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes). Diabetes mellitus associated with the mutation (mitochondrial diabetes) is a different phenotype from MELAS. We identified 11 patients with the mutation among 385 Japanese diabetic patients: two had MELAS and nine had mitochondrial diabetes. We present data on a male patient with mitochondrial diabetes who developed the nephrotic syndrome at the age of 23. Light microscopy revealed mesangial expansion, PAS-positive deposits and segmental sclerosis in the glomeruli. Scattered mesangial electron-dense deposits and thickening of the basement membrane were found on electron microscopy, suggesting that diabetic glomerulosclerosis accompanied by focal glomerulosclerosis (FGS). Mitochondrial diabetes may pre-dispose patients to renal complications, including forms of glomerulonephritis, such as FGS.

CDNAs for a Chymotrypsinogen-like Protein from Two Strains of Plodia interpunctella

Gut proteinases are involved in the solubilization and activation of insecticidal toxins produced by Bacillus thuringiensis and may also be involved in resistance development. Approximately threefold lower chymotrypsin-like enzyme activity was observed in a Bt( entomocidus)-resistant strain of the Indianmeal moth, Plodia interpunctella, than that in the Bt-susceptible strain. Because chymotrypsin-like proteinases are involved in Bt protoxin activation in P. interpunctella, we compared cDNA sequences, mRNA expression levels, and genomic DNA for chymotrypsin-like enzymes in Bt-susceptible and Bt-resistant strains of P. interpunctella. To isolate cDNA coding for chymotrypsinogen-like proteinases, a probe was developed using polymerase chain reaction (PCR) amplification of a cDNA library from the Bt-susceptible strain using a vector primer and a degenerate primer corresponding to a conserved sequence in the active site of serine proteinases. This probe was used to screen cDNA libraries from resistant and susceptible strains. Predicted amino acid sequences from cDNA clones of each strain share similarity with sequences of chymotrypsin-like proteinases and are most similar to a chymotrypsin-like proteinase from the tobacco hornworm, Manduca sexta. cDNAs for putative chymotrypsinogen-like proteins from both Bt-susceptible and Bt-resistant strains of P. interpunctella share an identical open reading frame of 846 nucleotides. The encoded proteins contain amino acid sequence motifs of serine proteinase active sites, disulfide-bridge cysteine residues, and both zymogen activation and signal peptides. A difference between these cDNAs was observed only in the untranslated region where a substitution of guanine for adenine occurred in the Bt-resistant strain. Southern and Northern blotting analyses indicated that there are no major differences in chymotrypsinogen-like genomic organization and mRNA expression in the two strains. These data suggest that chymotrypsinogen-like proteinase genes and their transcription are similar in the Bt-susceptible and Bt-resistant strains of P. interpunctella.

Specific labeling approaches to guanine and adenine imino and amino proton assignments in the AMP-RNA aptamer complex.

The secondary structure of a recently identified ATP-binding RNA aptamer consists of a purine-rich 11-residue internal loop positioned opposite a single guanine bulge flanked on either side by helical stem segments. The ATP ligand targets the internal loop and bulge domains, inducing a structural transition in this RNA segment on complex formation. Specifically, 10 new slowly exchanging proton resonances in the imino, amino and sugar hydroxyl chemical shift range are observed on AMP-RNA aptamer complex formation. This paper outlines site-specific labeling approaches to identify slowly exchanging imino (guanine) and amino (guanine and adenine) protons in internal loop and bulge segments of compact RNA folds such as found in the AMP-RNA aptamer complex. One approach incorporates 15N-labeled guanine (N1 imino and N2 amino positions) and 15N-labeled adenine (N6 amino position), one residue at a time, in the AMP-binding RNA aptamer, with labeling incorporation through chemical synthesis facilitated by generating the aptamer from two separate strands. The unambiguous assignments deduced from the 15N labeling studies have been verified from an independent labeling strategy where individual guanines in the internal loop have been replaced, one at a time, by inosines and assignments were made on the basis of the large 2 ppm downfield shift of the guanine imino protons on inosine substitution. The strengths and limitations of the inosine-for-guanine substitution approach emerge from our studies on the AMP-RNA aptamer complex. The assignment of the internal loop and bulge imino and amino protons was critical in our efforts to define the solution structure of the AMP-RNA aptamer complex since these slowly exchanging protons exhibit a large number of long-range intramolecular NOEs within the RNA, as well as intermolecular NOEs to the AMP in the complex. The current application of specific 15N and inosine labeling approaches for exchangeable imino and imino proton assignments in the nonhelical segments of an RNA aptamer complex in our laboratory complements selective 2H and 13C approaches to assign nonexchangeable base and sugar protons in RNA and ligand-RNA complexes reported in the literature.

Lack of expansion of cytosine adenine guanine trinucleotide repeat in the dentatorubral and pallidoluysian atrophy gene in swedish schizophrenic patients

Hereditary dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder. It is characterized by a combination of progressive myoclonus, epilepsy, ataxia, choreoathetosis, and dementia. In a few cases schizophrenic symptoms have also been observed preceding the DRPLA diagnosis (Nakano et al 1985; Naito et al 1987). Recently DRPLA has been associated with expanded cytosine adenine guanine (CAG) trinucleotide repeats in a gene (B37; Li et al 1993) on chromosome 12p in several Japanese (Koide et al 1994; Nagafuchi et al 1994) and two European families (Warner et al 1994). One patient lacking family history for DRPLA was also found to have expanded trinucleotide repeats within gene B37 (Koide et al 1994). The reports of schizophrenic symptoms among DRPLA cases prompted us to investigate unrelated Swedish patients (n = 118) fullfilling DSM-III-R (American Psychiatric Association 1987) criteria for schizophrenia. Diagnoses were based on clinical interviews and reviews of medical records as previously described (Jrnsson et al 1993). Venous blood was taken from all individuals into EDTA-containing tubes. DNA was isolated

A Novel Point Mutation (R243Q) in Exon 7 of the c-erbAβ Thyroid Hormone Receptor Gene in a Family with Resistance to Thyroid Hormone

Resistance to thyroid hormone (RTH) is characterized by variable tissue hyporesponsiveness to thyroid hormones. Recently, a large number of different point mutations have been identified in the c-erbA beta thyroid hormone receptor (TR beta) in subjects with RTH. We describe a Japanese family with RTH with a novel point mutation in exon 7 of the TR beta gene. A single nucleotide substitution, guanine for adenine, was identified at the second position of codon 243 located in the hinge domain between the ligand binding and DNA binding domains in one of the two alleles of the proband and his mother, resulting in the substitution of the normal arginine (CGG) with a glutamine (CAG). Except for one family, point mutations so far described in RTH are clustered at exons 8-10 of the TR beta gene. This report presents a novel mutation in the characteristic portion in exon 7 of the TR beta.

Sudden cardiac death in hypertrophic cardiomyopathy. Variability in phenotypic expression of beta-myosin heavy chain mutations.

Recent identification of mutations in the beta-myosin heavy chain gene (MYH7), a major responsible gene for HCM, has provided the opportunity to characterize genotype-phenotype correlation in HCM families. In this study we analysed the phenotypic expression of two beta-myosin heavy chain (beta MHC) mutations in three unrelated HCM families. Living individuals from three unrelated HCM families (Families 1, 2, and 3) were screened by history, physical examination, electrocardiography, and two-dimensional echocardiography. Blood was collected from all individuals for DNA extraction. Polymerase chain reaction (PCR), restriction endonuclease digestion and chemical cleavage were utilized for detection of mutations. All mutations were confirmed by sequence analysis. Identification of mutations: A missense mutation in exon 13 of the beta MHC gene (Arg403 Gln) was detected in HCM patients from Families 1 and 2. PCR amplification of the exon 13 DNA, followed by Ddel digestion of the PCR product and gel electrophoresis, showed two fragments of 84 and 70 bp in normal individuals and four fragments of 84, 70, 52 and 32 bp in HCM patients. Sequence analysis showed substitution of an adenine for guanine at coding position 1208. In Family 3, a missense mutation in exon 16 of the beta MHC gene (Val606 Met) was detected in HCM patients. Chemical cleavage of the PCR products showed an uncleaved product of 337 bp in the normal individuals, while in the affected individuals, in addition to the uncleaved product, a 90 bp cleaved product was also detected, indicating the presence of a mismatch in one allele. Sequence analysis showed substitution of an adenine for guanine in coding position 1817. Seven members of Family 1 had HCM, of whom five are alive. One patient died from sudden cardiac death (SCD) and another from recurrent cerebral emboli. In Family 2, 15 individuals had HCM of whom nine have died, seven from SCD. The mean age at the time of SCD was 33 years. The third family is comprised of 11 affected individuals and one obligate carrier, of whom one patient died at age 17 from progressive heart failure. Two additional individuals in this family have also succumbed to SCD to age 60. A variety of clinical and echocardiographic manifestations of HCM were present in each family. Logrank test of Kaplan-Meier survival curves indicates that Arg403 Gln mutation was associated with a poor prognosis in HCM families as compared to Val606 Met (P = 0.034). beta MHC mutations despite showing variable clinical and echocardiographic manifestations of HCM are predictors of survival in HCM families.

A Subtype of Diabetes Mellitus Associated with a Mutation of Mitochondrial DNA

Several families have been described in which a mutation of mitochondrial DNA, the substitution of guanine for adenine (A-->G) at position 3243 of leucine transfer RNA, is associated with diabetes mellitus and deafness. The prevalence, clinical features, and pathophysiology of diabetes with this mutation are largely undefined. We studied 55 patients with insulin-dependent diabetes mellitus (IDDM) and a family history of diabetes (group 1), 85 patients with IDDM and no family history of diabetes (group 2), 100 patients with non-insulin-dependent diabetes mellitus (NIDDM) and a family history of diabetes (group 3), and 5 patients with diabetes and deafness (group 4) for the mutation. We also studied the prevalence and characteristics of diabetes in 39 patients with a syndrome consisting of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes who were known to have the mutation and 127 of their relatives (group 5). We identified 16 unrelated patients with diabetes associated with the A-->G mutation: 3 patients from group 1 (6 percent), 2 patients from group 3 (2 percent), 3 patients from group 4 (60 percent), and 8 patients from group 5 (21 percent). We also identified 16 additional subjects who had diabetes and the mutation among 42 relatives of the patients with diabetes and the mutation in groups 1, 2, 3, and 4 and 20 affected subjects among the 127 relatives of the patients in group 5. Diabetes cosegregated with the mutation in a fashion consistent with maternal transmission, was frequently (in 61 percent of cases) associated with sensory hearing loss, and was generally accompanied by impaired insulin secretion. Diabetes mellitus associated with the A-->G mutation at position 3243 of mitochondrial leucine transfer RNA represents a subtype of diabetes found in both patients with IDDM and patients with NIDDM in Japan.

Familial Defective Apolipoprotein B-100 Is Clinically Indistinguishable From Familial Hypercholesterolemia

Familial defective apolipoprotein B-100 is caused by a substitution of adenine for guanine in exon 26 of the gene coding for apolipoprotein B, which results in the substitution of glutamine for arginine in the putative low-density lipoprotein-receptor binding domain of the mature protein. This amino acid substitution diminishes the binding capacity of the low-density lipoprotein particle for the low-density lipoprotein receptor, which in turn leads to an increase in levels of plasma total and low-density lipoprotein cholesterol. To identify carriers of this mutation by means of molecular biology techniques in a large cohort of Dutch patients living in the Netherlands and in Canada with primary hypercholesterolemia, to establish the frequency of the disorder, and to investigate its clinical signs and symptoms and the response to cholesterol-lowering therapy. A total of 1248 patients were screened, and the mutation was found in 18 patients who were initially all diagnosed as having familial hypercholesterolemia. Ten of 18 patients had tendon xanthomas or an arcus cornealis or both, and eight of 18 patients had angina or other evidence of coronary artery disease. The disorder was clinically indistinguishable from familial hypercholesterolemia in terms of physical characteristics and lipoprotein measures. Response to lipid-lowering therapy with beta-hydroxy-beta-methylglutaryl coenzyme A reductase inhibitors was similar to that reported in patients with familial hypercholesterolemia. The mutation was associated with a similar haplotype, which was also reported in other patients of Western European descent with familial defective apolipoprotein B100. This strongly suggests that the mutation has a common chromosomal background that originated in Western Europe.

Molecular genetic heterogeneity of myophosphorylase deficiency (McArdle's disease).

Myophosphorylase deficiency (McArdle's disease) is one of the most common causes of exercise intolerance, muscle cramps, and recurrent myoglobinuria. The myophosphorylase gene has been sequenced and assigned to chromosome 11, but the molecular basis of McArdle's disease is not known. We sequenced complementary DNA in 4 patients and studied genomic DNA by restriction-endonuclease analysis in 40 patients with McArdle's disease. Sequence analysis revealed three distinct point mutations: the substitution of thymine for cytosine at codon 49 in exon 1, changing an encoded arginine to a stop codon; the substitution of adenine for guanine at codon 204 in exon 5, changing glycine to serine; and the substitution of cytosine for adenine at codon 542 in exon 14, changing lysine to threonine. Analysis of restriction-fragment-length polymorphisms of appropriate fragments of genomic DNA after amplification with the polymerase chain reaction showed that 18 patients were homozygous for the stop-codon mutation, 6 had different mutations in the two alleles (compound heterozygotes), and 11 were presumed to be compound heterozygotes for a known mutation and an unknown one; only 5 patients had none of the three mutations. All three mutations were present in various combinations in five members of a family in which transmission appeared to be autosomal dominant. McArdle's disease is genetically heterogeneous, but the most common mutation is the substitution of thymine for cytosine at codon 49. These results suggest that in about 90 percent of patients the diagnosis of McArdle's disease can be made from a patient's leukocytes, thus avoiding the need for muscle biopsy.

Familial amyloidosis, Finnish type: G654 → A mutation of the gelsolin gene in Finnish families and an unrelated American family

The Finnish type of familial amyloid polyneuropathy (FAF) is an autosomal dominant form of systemic amyloidosis caused by a mutation in the gelsolin gene. The mutation leads to the expression of amyloidogenic mutant Asp187 → Asn gelsolin, an actin-modulating protein. We previously developed a DNA test based on amplification by the polymerase chain reaction followed by allele-specific oligonucleotide hybridization that identifies the base substitution adenine for guanine at nucleotide 654 in the gelsolin gene causing the disease. We show here that the same mutation is present in members of six apparently unrelated Finnish families and in a member of an unrelated American family. These results, taken together with previously published findings in nine additional Finnish families and another unrelated American family, indicate that most, perhaps all, FAF patients in Finland and possibly worldwide carry the same mutation. We suggest two alternative explanations: (i) the mutation arose in a very early common ancestor or (ii) the Asn187 mutation is particularly, perhaps uniquely, amyloidogenic.

Molecular basis of lipoprotein lipase deficiency in two Austrian families with type I hyperlipoproteinemia

To determine the molecular basis for type I hyperlipoproteinemia in two Austrian families, the lipoprotein lipase (LPL) gene of two patients exhibiting LPL deficiency was analyzed by Southern blotting and by direct genomic sequencing of DNA amplified by polymerase chain reaction (PCR). All exons of the LPL gene except part of the noncoding region of exon 10, all splice donor and acceptor sites, as well as 430 basepairs of the 5′-region including the promotor were sequenced. A homozygous substitution of adenine for guanine in the fifth exon at cDNA position 818 of the LPL gene was found in both patients. Our sequencing strategy largely ruled out a linkage disequilibrium of the identified nucleotide change with another defect potentially causing the clinical phenotype. The base change described abolishes a normally present AvaII restriction site allowing the identification of carriers of the mutant allele by AvaII digestion of PCR fragments of exon 5; three members of the two families were homozygous for this mutation and ten members were heterozygous. The activity of LPL in postheparin plasma was almost completely absent in homozygotes and about half normal in heterozygotes. The loss of activity was related to LPL protein structure. This mutation alters the amino acid sequence at residue 188 from Gly to Glu. The conformational preferences of the protein chain around position 188 were calculated with the use of a knowledge-based computerized method. The most probable conformation is a beta-turn formed by residues 189–192. The mutation seems to destabilize the beta-turn and/or a yet larger domain critical for substrate alignment.

Mutation in gelsolin gene in Finnish hereditary amyloidosis.

Familial amyloidosis, Finnish type (FAF), is an autosomal dominant form of familial amyloid polyneuropathy. The novel amyloid fibril protein found in these patients is a degradation fragment of gelsolin, an actin-binding protein. We found a mutation (adenine for guanine) at nucleotide 654 of the gelsolin gene in genomic DNA isolated from five FAF patients. This site is polymorphic since the normal allele was also present in all the patients tested. This mutation was not found in two unaffected family members and 11 normal controls. The A for G transition causes an amino acid substitution (asparagine for aspartic acid) that was found at position 15 of the amyloid protein. The mutation and consequent amino acid substitution may lead to the development of FAF.

A point mutation in transthyretin increases affinity for thyroxine and produces euthyroid hyperthyroxinemia.

In a family expressing euthyroid hyperthyroxinemia, an increased association of plasma thyroxine (T4) with transthyretin (TTR) is transmitted by autosomal dominant inheritance and is secondary to a mutant TTR molecule with increased affinity for T4. Eight individuals spanning three generations exhibited the abnormality. Although five of eight individuals had elevated total T4 concentrations, all affected individuals were clinically euthyroid and all had normal free T4 levels. Purified TTR from the propositus had an affinity for 125I-T4 three times that of control TTR. Exons 2, 3, and 4 (representing greater than 97% of the coding sequence) of the TTR gene of DNA prepared from the propositus' peripheral blood leukocytes were amplified using the polymerase chain reaction (PCR) and were sequenced after subcloning. Exons 2 and 3 were indistinguishable from normal. In 50% of clones amplified from exon 4, a substitution of adenine (ACC) for guanine (GCC) in codon 109 resulted in the replacement of threonine-for-alanine, a mutation confirmed by amino acid sequencing of tryptic peptides derived from purified plasma TTR. The adenine-for-guanine substitution abolishes one of two Fnu 4H I restriction sites in exon 4. PCR amplification of exon 4 of TTR and restriction digestion with Fnu 4H I confirmed that five affected family members with increased binding of 125I-T4 to TTR are heterozygous for the threonine 109 substitution that increases the affinity of this abnormal TTR for T4.

Identification of the crossing-over point of a hybrid gene encoding human glycophorin variant Sta. Similarity to the crossing-over point in haptoglobin-related genes.

One of the human glycophorin variants, Stones (Sta), has been shown to be the product of a hybrid gene of which the 5'-half derived from the glycophorin B (GPB) gene whereas the 3'-half derived from the glycophorin A (GPA) gene. The present study reveals the crossing-over point of this hybrid gene from the analysis of polymerase chain reaction products. The genomic sequences encompassing the region corresponding to exon 3 to exon 4 of GPA were amplified by polymerase chain reaction with oligonucleotide primers synthesized according to GPA and GPB genomic sequences (Kudo, S., and Fukuda, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4619-4623). After subcloning the products, the nucleotide sequences derived from GPA, GPB, and putative Sta genes were determined. Comparison of the nucleotide sequences of GPA, GPB, and Sta genes indicate that the crossing-over took place 200 base pairs upstream from the first nucleotide of exon 4. Intriguingly, the nucleotide sequence surrounding the putative crossing-over point is homologous to the crossing-over point proposed for haptoglobin genes (Maeda, N., McEvoy, S.M., Harris, H.F., Huisman, T.H.J., and Smithies, O. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7395-7399). These results suggest strongly that homologous recombination through unequal crossing-over can be facilitated by specific genomic elements, such as those in common in these two crossing-over events. The present study also revealed that this Sta individual has a variant GPA gene; substitution of adenine for guanine at the nucleotide for codon 39 results in substitution of lysine for arginine at amino acid 39, and loss of an SstI restriction site.

Use of host cell reactivation of cisplatin-treated adenovirus 5 in human cell lines to detect repair of drug-treated DNA.

This study demonstrates that whilst some DNA-repair deficiencies can be detected using host cell reactivation of cisplatin (CDDP)-treated adenovirus (Ad5), not all repair deficiencies affected replication of CDDP-treated Ad5 in human cells. A line of fibroblasts (XP25), derived from a patient with a UV-hypersensitive syndrome xeroderma pigmentosum (XP), was found, as previously reported [1], to be deficient in reactivating the treated virus when compared to the apparently repair-proficient human tumor cell lines established from bladder and ovarian carcinomas. However, a testicular teratoma cell line (SuSa), shown previously to be deficient in the repair of guanine-guanine (G-G) intrastrand crosslinks, adenine-guanine (A-G) intrastrand crosslinks and interstrand crosslinks [2], was found to reactivate the treated virus to a similar extent as the repair-proficient ovarian tumor cell line and the similarly repair-proficient RT112 cell line derived from a bladder carcinoma. Therefore, not all repair-deficient cell lines were deficient at CDDP-treated Ad5 reactivation. However, the HCR technique may still prove to be useful as a rapid screen for DNA-repair deficiencies in CDDP-sensitive cells of unknown repair capacity. A CDDP-sensitive ovarian tumor cell line (TR175) was deficient in reactivating CDDP-treated Ad5, whilst another ovarian cell line (TR170) of intermediate CDDP sensitivity reactivated the virus to a marginally higher extent than the other more CDDP-resistant repair proficient ovarian cell line (SKOV3). In addition, sublines of either the SuSa cell or the RT112 cells expressing approximately two-fold levels of resistance or increased sensitivity to CDDP, showed no change in their abilities to reactivate this CDDP-treated virus, compared to their parental lines. CDDP-treated Ad5 was also used as a lethal probe to obtain cell lines specifically deficient in DNA repair. One such deficient line (SKOV3-C3A), derived from the SKOV3 ovarian carcinoma cell line, displayed an unusual biphasic curve for reactivation of the CDDP-treated virus. Further cell lines derived in this novel manner may prove useful in analysing the genetics of CDDP-repair.

Matrix-isolation of H·-induced Free Radicals from Purines in Acidic Glasses

H.(D.)-atoms produced by photo-oxidation (lambda = 254 nm) of Fe2+ in acidic glasses (6 mol dm-3 H2SO4/H2O or D2SO4/D2O) at 77K were allowed to react with the purine bases adenine, guanine, hypoxanthine and xanthine as well as with ribo- and deoxyribosides (-tides) of adenine and guanine by annealing to 110-130 K. The ensuing free radicals were studied by electron spin resonance (ESR) spectroscopy at 77 K. Individual radical species were assigned by simulation of patterns isolated from thermal annealing studies up to 180 K. It is shown that H.-atom reaction with the bases produces C2- and C8-addition radicals for adenine and C8-addition species for guanine. Guanine is also photo-oxidized directly in the glass, producing a cation or its deprotonated successor species. In the nucleosides (-tides) of both bases, H. atoms were found to abstract hydrogen from carbon sites C1', C2' or C3', and C5' for ribosides and C1', C2', and C5' for deoxyribosides (-tides), respectively.

Single base mutation in the pro alpha 2(I) collagen gene that causes efficient splicing of RNA from exon 27 to exon 29 and synthesis of a shortened but in-frame pro alpha 2(I) chain.

Previous observations demonstrated that a lethal variant of osteogenesis imperfecta had two altered alleles for pro alpha 2(I) chains of type I procollagen. One mutation produced a nonfunctioning allele in that there was synthesis of mRNA but no detectable synthesis of pro alpha 2(I) chains from the allele. The mutation in the other allele caused synthesis of shortened pro alpha 2(I) chains that lacked most or all of the 18 amino acids encoded by exon 28. Subclones of the pro alpha 2(I) gene were prepared from the proband's DNA and the DNA sequence was determined for a 582-base-pair (bp) region that extended from the last 30 bp of intervening sequence 26 to the first 26 bp of intervening sequence 29. Data from six independent subclones demonstrated that all had the same sequence as a previously isolated normal clone for the pro alpha 2(I) gene except that four subclones had a single base mutation at the 3' end of intervening sequence 27. The mutation was a substitution of guanine for adenine that changed the universal consensus sequence for the 3' splicing site of RNA from -AG- to -GG-. S1 nuclease experiments demonstrated that about half the pro alpha 2(I) mRNA in the proband's fibroblasts was abnormally spliced and that the major species of abnormal pro alpha 2(I) mRNA was completely spliced from the last codon of exon 27 to the first codon of exon 29. The mutation is apparently unique among RNA splicing mutations of mammalian systems in producing a shortened polypeptide chain that is in-frame in terms of coding sequences, that is used in the subunit assembly of a protein, and that contributes to a lethal phenotype.