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PubMed1996

Genetic bases of human complement C7 deficiency.

Complement C7 deficiency (C7D) is associated frequently with recurrent bacterial infections, especially meningitis caused by Neisseria meningitidis. We report in this work the molecular bases of C7D in two unrelated Japanese males. We used exon-specific PCR/single-strand conformation polymorphism analysis as a screening step for mutations. Subsequent direct sequencing of the target exons identified homozygous mutations in exon 16 of case 1 and in exon 15 of case 2. The mutation of case 1 was a homozygous T to A transversion at nucleotide 2250, the third nucleotide of the codon TGT for Cys728, leading to a stop codon TGA (C728X). In case 2, a homozygous 2-bp deletion (2137delTG/2138delGT/2139delTG) caused a frameshift, generating a premature termination codon 4 to 6 nucleotides downstream. Family study in case 1 confirmed the genetic nature of the defect. Moreover, we detected a novel polymorphism in intron 11 that presumably is linked to the mutation responsible for C7D in case 1. Our results indicate that the pathogenesis of C7D is heterogeneous like most of the other deficiencies of complement components.

Adult↗
PubMed1996

A novel nonsense mutation (G181X) in the human cholesteryl ester transfer protein gene in Japanese hyperalphalipoproteinemic subjects.

Cholesteryl ester transfer protein (CETP) plays an important role in regulating the concentration and composition of high density lipoprotein (HDL) and low density lipoprotein (LDL). Although several genetic abnormalities causing CETP deficiency have been identified in the Japanese subjects with a marked hyperalphalipoproteinemia (HALP), there are many CETP-deficient subjects for whom the genetic abnormalities have not been clarified. In the present study, we analyzed the molecular basis of an HALP subject without CETP activity and mass, and found a novel mutation in the CETP gene. This novel mutation (G181X) was a G-to-T substitution at codon 181 of exon 6 which replaced a codon for glycine (GGA) with a premature stop codon (TGA). The G181X mutation created a new cutting site by restriction enzyme MaeIII. To estimate the frequency of G181X, we investigated unrelated 294 HALP (HDL-cholesterol > or = 2.59 mmol/L = 100 mg/dl) subjects by restriction fragment length polymorphism (RFLP) analysis with Mae III. One (0.34%) HALP subject was homozygous and four (1.36%) were heterozygous for this mutation. The allelic frequency of a G-to-T substitution at codon 181 of exon 6 was 0.0102 in HALP subjects. From the lipid analysis of the proband and the homozygote, it was clarified that the G181X mutation had dominant effects on HDL and LDL metabolism, similar to a G-to-A substitution at the 5' splice donor site of the intron 14 (1451 + 1G-->A). In conclusion, the G181X mutation is one of causes of HALP in the Japanese HALP subjects, having dominant effects on lipid metabolism.

Carrier Proteins↗
PubMed1998

Genetic basis of human complement C8 alpha-gamma deficiency.

Deficiency of the alpha-gamma subunit of the eighth component of complement (C8alpha-gammaD) is frequently associated with recurrent neisserial infections, especially meningitis caused by Neisseria meningitidis. We here report the molecular basis of C8alpha-gammaD in two unrelated Japanese subjects. Screening all 11 exons of the C8alpha gene and all 7 exons of the C8gamma gene and their boundaries by exon-specific PCR/single-strand conformation polymorphism demonstrated aberrant single-stranded DNA fragments in exon 2 of C8alpha gene in case 1 and in exons 2 and 9 of C8alpha gene in case 2. Nucleotide sequencing of the amplified DNA fragments in case 1 revealed a homozygous single-point mutation at the second exon-intron boundary, inactivating the universally conserved 5' splice site consensus sequence of the second intron (IVS2+1G-->T). Case 2 was a compound heterozygote for the splice junction mutation, IVS2+1G-->T, and a nonsense mutation at Arg394 (R394X). R394X was caused by a C to T transition at nucleotide 1407, the first nucleotide of the codon CGA for Arg394, leading to a stop codon TGA. No mutations were detected in the C8gamma gene by our method. Our results indicate that the pathogenesis of C8alpha-gammaD might be caused by heterogeneous molecular defects in the C8alpha gene.

Adult↗
PubMed1994

Hyperphenylalaninemia due to defects in tetrahydrobiopterin metabolism: molecular characterization of mutations in 6-pyruvoyl-tetrahydropterin synthase.

A variant type of hyperphenylalaninemia is caused by a deficiency of tetrahydrobiopterin (BH4), the obligatory cofactor for phenylalanine hydroxylase. The most frequent form of this cofactor deficiency is due to lack of 6-pyruvoyl-tetrahydropterin synthase (PTPS) activity, the second enzyme in the biosynthetic pathway for BH4. The human liver cDNA for PTPS was previously isolated, and the recombinant protein was found to be active when expressed in Escherichia coli. We now have investigated two patients for their molecular nature of this autosomal recessive disorder. Both patients were diagnosed as PTPS deficient, one with the central and one with the peripheral form, on the basis of an elevated serum phenylalanine concentration concomitant with lowered levels of urinary biopterin and PTPS activity in erythrocytes. Molecular analysis was performed on the patients' cultured primary skin fibroblasts. PTPS activities were found in vitro to be reduced to background activity. Direct cDNA sequence analysis using reverse transcriptase-PCR technology showed for the patient with the central from a homozygous G-to-A transition at codon 25, causing the replacement of an arginine by glutamine (R25Q). Expression of this mutant allele in E. coli revealed 14% activity when compared with the wild-type enzyme. The patient with the peripheral form exhibited compound heterozygosity, having on one allele a C-to-T transition resulting in the substitution of arginine 16 for cysteine (R16C) in the enzyme and having on the second allele a 14-bp deletion (delta 14bp), leading to a frameshift at lysine 120 and a premature stop codon (K120-->Stop). Heterologous expression of the enzyme with the single-amino-acid exchange R16C revealed only 7% enzyme activity, whereas expression of the deletion allele delta 14bp exhibited no detectable activity. All three mutations, R25Q, R16C, and K120-->Stop, affect evolutionarily conserved residues in PTPS, result in reduced enzymatic activity when reconstituted in E. coli, and are thus believed to be the molecular cause for the BH4 deficiency. This is the first report describing mutations in PTPS that lead to BH4 deficiency.

Alcohol Oxidoreductases↗
PubMed1999

Compound heterozygosity for one novel and one recurrent mutation in a Thai patient with severe protein S deficiency.

Homozygous or compound heterozygous protein S (PS) deficiency is a very rare disorder in the anticoagulant system, that can lead to life-threatening thrombotic complications shortly after birth. This report describes the results of the genetic analysis of the PROS 1 genes in a Thai girl patient. She was reported in 1990 as the first case with homozygous PS deficiency and neonatal purpura fulminans. In the present report, we identified the mutations in this patient by direct sequencing of PCR products representing all 15 exons of the PROS 1 gene and their flanking intronic regions. The patient turned out to be compound heterozygous for two null mutations. One allele contained a novel sequence variation, an A-insertion in an A5-tract covering codon 146 and 147, that results in a frameshift and a stop codon (TAA) at position 155. The other allele contained a nonsense mutation in exon 12 by a transition at codon 410 CGA (Arg) to TGA (stop). Cosegregation of PS deficiency with these two genetic defects was observed in her family.

Alleles↗
PubMed1990

Expression of a ciliate gene in Escherichia coli using a suppressor tRNA to read the UAA and UAG glutamine codons.

Most ciliates use a particular genetic code where the standard stop codons UAA and UAG encode glutamine. Ciliate genes cannot therefore be expressed in heterologous systems such as Escherichia coli. To overcome this problem, we worked out a system of inducible suppression to permit efficient readthrough of UAAs and UAGs: a strong UAA tRNA suppressor that inserts glutamic acid was cloned downstream from a tac promoter whose efficiency was reduced by a transcription terminator. This system proved to be operational (1) to suppress UAG mutations by wobble pairing in an E. coli lacI-lacZ gene fusion and (2) to read through at least eight UAA glutamine codons in a Paramecium alpha-tubulin gene, as detected by Western blotting and colony hybridization. This work opens the way for cloning Ciliate genes from expression libraries and for expressing particular sequences without extended in vitro mutagenesis. A similar approach can be envisaged for expression of genes from Mycoplasma, mitochondria or other genomes that use non-standard genetic codes.

Animals↗
PubMed2000

Combined enzymatic complex I and III deficiency associated with mutations in the nuclear encoded NDUFS4 gene.

Combined OXPHOS-system enzyme deficiencies are observed in approximately 25% of all OXPHOS-system disturbances. Of these, combined complex I and III deficiency is relatively scarce. So far, only mtDNA and thymidine phosphorylase (TP) mutations have been associated with combined OXPHOS-system disturbances. In this report we show, for the first time, that a nuclear gene mutation in a structural, nuclear encoded complex I gene is associated with combined complex I and III deficiency. After our initial report we describe mutations in the NDUFS4 gene of complex I in two additional patients. The first mutation is a deletion of G at position 289 or 290. Amino acid 96 changes from a tryptophan to a stop codon. The mutation was found homozygous in the patient; both parents are heterozygous for the mutation. The second mutation is a transition from C to T at cDNA position 316. Codon is changed from CGA (arginine) to TGA (stop). The patient is homozygous for the mutation; both parents are heterozygous. Both mutations in the NDUFS4 gene led to a premature stop in Leigh-like patients with an early lethal phenotype. We hypothesise that the structural integrity of the OXPHOS system, in mammal supermolecular structures, may be responsible for the observed biochemical features.

Base Sequence↗
PubMed2001

A frameshift mutation in the human fibrinogen Aalpha-chain gene (Aalpha(499)Ala frameshift stop) leading to dysfibrinogen San Giovanni Rotondo.

We have investigated a 53-yr-old asymptomatic white man with decreased functional, but not immunologic, fibrinogen plasma levels together with prolonged thrombin and reptilase times, detected through routine coagulation studies prior to a surgical procedure. A new heterozygous single nucleotide deletion (C) at position Ala499 within the Aalpha-chain gene was identified, which predicted changes of the corresponding amino acids encoded by the subsequent portion of the exon V and the appearance of a premature stop codon at position 518 (Aalpha[499]Ala frameshift stop). The new dysfunctional fibrinogen, San Giovanni Rotondo variant, was confirmed in vivo by SDS-PAGE analysis of HPLC-purified fibrinogen chains. Mass spectrum examination of the abnormal HPLC-purified peak gave an estimated mass (56,088 Da) similar to that predicted by DNA analysis of the mutated Aalpha-chain gene (56,088 Da) and, after tryptic digestion, the truncated Aalpha-chain was shown only in the propositus, who also carried normal Aalpha-chain. In addition, mass spectrum analysis of the tryptic digest of the abnormal chain confirmed the presence of a new and unpaired cysteine at the last position that was predicted to form a disulfide bridge with human serum albumin. Immuno-blot analysis confirmed that fibrinogen San Giovanni Rotondo variant, but not normal fibrinogen. contained substantial amounts of albumin. Present findings confirm that truncated Aalpha-chain lacking part of the terminal domain may be incorporated into mature fibrinogen molecules and normally secreted in the bloodstream.

Afibrinogenemia↗
PubMed2006

Three novel point mutations of the CYP21 gene detected in classical forms of congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) [OMIM 201 910] is a group of autosomal recessive disorders most commonly due to 21-hydroxylase deficiency and presenting with a wide range of clinical manifestations. A limited number of inactivating pseudogene-derived mutations account for the majority of 21-hydroxylase gene ( CYP21) mutations, additional rare mutations can be found in single families and small populations. We found three novel CYP21 mutations in CAH patients suffering from the classical form of the disease, of which one is a frameshift mutation (1353-1354insA) leading to a premature termination codon (K277K, Q228A...E294X), one results in a premature stop codon (2551C>T, R444X), and one is a missense mutation (2609T>C; P463L). The frameshift and premature stop mutations can be predicted to result in a CYP21 protein without any residual enzyme activity. To determine the functional consequences of the P463L mutation, the IN VITRO enzyme activity was studied in COS-7 cells and revealed a reduced 21-hydroxylase activity of 2.6+/-0.8 (SD)% for the conversion of 17-hydroxyprogesterone (17OHP) to 11-deoxycortisol and of 3.0+/-0.5 % for the conversion of progesterone to 11-deoxycorticosterone (DOC). We conclude that functional analyses of unknown mutations provide information on the disease severity and should be always performed when novel CYP21 mutations are detected. Knowledge of the residual 21-hydroxylase function improves both genetic counselling and individual clinical management in CAH patients.

Adrenal Hyperplasia, Congenital↗
PubMed1996

Differential expression of the heavy-chain ferritin gene in non-adhered and adhered oligodendrocytes.

Oligodendrocytes (OLGs) synthesize and maintain central nervous system myelin. Little is known about the molecules and pathways involved in signaling the commencement of myelination; yet myelination is spatially and temporally controlled. We are interested in deciphering the signaling events that control the on and off switch of myelination. To address these questions we are using an in vitro model system consisting of pure cultures of OLGs, isolated from postmyelination brains. We have shown that in vitro, these OLGs regenerate and reenact the ontogenic development of myelin upon adhesion to a substratum; we have also shown that when transplanted in vivo, they can form myelin. Hence, identifying the genes that are turned on upon OLG adhesion seemed a worthwhile approach to singling out those genes that are critical to the differentiation process. For this we adopted a novel technique-differential display-that permits the comparison of gene expression under two sets of conditions; in our case between non-adhered and adhered OLGs. Using the differential display method in conjunction with a set of five arbitrary primers, we have identified five cDNAs that are activated upon OLG substratum adhesion. Four of these cDNAs proved to be fragments of the heavy (H)-chain ferritin cDNA. H-chain ferritin is involved in the rapid sequestration and delivery of iron; it is also a cytoprotectant. Brain iron is localized predominantly in OLGs. Our finding that the expression of H-chain ferritin is upregulated upon OLG regeneration/differentiation agrees with reports, in other cell types, that H-chain ferritin transcription is modulated by factors that control cell growth and differentiation. The enhanced transcription results in a twofold augmentation in ferritin synthesis. This is the first demonstration of an adherence-mediated activation of the H-chain ferritin gene. The observation has interesting implications. The transcription of the H-chain ferritin gene is associated with tissue stress such as might occur during rapid cell growth. The fact that this transcription takes place upon OLG-substratum adhesion can be viewed as another manifestation of the anchorage-induced signal that drives OLGs toward a differentiation program. This entails the synthesis of all the necessary components for the assembly of large quantities of membranes, hence constituting a period of rapid growth and considerable cell stress. We have sequenced the full-length ferritin cDNA of ovine OLGs; the cDNA is 775 bp long. The coding sequence starts at residue 57 with the ATG codon and terminates at residue 602, with the stop codon TAA. The cDNA codes for 181 amino acids with a predicted size for the protein of 22 kD. The deduced amino acid sequence is 93% identical to human ferritin. We postulate that OLG H-chain ferritin may function at more than one level: 1) it provides the iron required to sustain an oxidative metabolism; and 2) it acts as a cytoprotectant against oxidant-mediated injury.

Amino Acid Sequence↗
PubMed1998

Four new mutations in the DNA mismatch repair gene MLH1 in colorectal cancers with microsatellite instability. Mutations in brief no. 157. Online.

Hereditary nonpolyposis colorectal cancer (HNPCC) is frequently associated with inherited mutation in one of four DNA mismatch repair genes. Somatic mutations in the same genes are also found in a subset of sporadic colorectal cancers. A defect in DNA mismatch repair results in a RER (replication error) tumor phenotype. We screened 110 archival and 11 prospectively acquired colorectal cancers for the RER phenotype. A total of 22 cancers were RER-positive. RER-positive tumors were investigated for mutations in the DNA mismatch repair gene MLH1 using single-strand-conformation-polymorphism (SSCP) analysis. We identified four previously undescribed mutations in four different samples. Three mutations were exonic: a point mutation at codon 69 (AGG-->AAG(arg-->lys]); a single base pair deletion at codon 42/43 (GCAAAATCC-->GCAAATCC) leading to a new stop codon downstream; and a point mutation at codon 757 (TAA-->TAT [termination-->tyr] which extend the MLH1 peptide by 36 ammino acids. The fourth mutation was a 1 base pair insertion six base pairs 5' to the start of exon 14 (tttgtttt-->tttggtttt). The mutations were not seen in the patients' constitutional DNA. The somatic MLHI mutations identified appear to be causally associated with the RER phenotype.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Molecular organization of the glutathione reductase gene in Drosophila melanogaster.

Glutathione reductase catalyzes the conversion of the oxidized form of glutathione to regenerate reduced glutathione, which acts as a versatile intracellular reductant. The present study provides initial characterization of the glutathione reductase gene in Drosophila melanogaster and its response to experimentally induced oxidative stress. Drosophila cDNA clones were isolated, based on cross-hybridization to the Musca domestica glutathione reductase cDNA. Genomic clones were isolated by cross-hybridization with the Drosophila cDNA as hybridization probe. Northern analysis of adult Drosophila poly(A)+ RNA, utilizing the Drosophila cDNA probe, revealed a hybridization signal in the 2-kb range. The entire sequence of one cDNA was determined. In addition to a coding domain of 1431 bases, the sequence included 206 bases upstream of a putative start codon and 355 bases downstream of a putative stop codon. Based on the cDNA sequence, the 476 amino acid sequence of the Drosophila glutathione reductase gene was deduced and was found to have extensive similarities with the glutathione reductase gene from other species. Gene mapping of a 13-kb genomic fragment revealed that the glutathione reductase gene consists of at least two exons spanning approximately 5 kb. A first exon contains sequence for only the first 5 amino acids and the first base of the sixth and appears to be separated by a ca. 2.5-kb intron from the remainder of the coding region, which is confined to <2 kb. The Drosophila glutathione reductase is single copy and its cytogenetic position, as determined by in situ hybridization, is 7D-E on the X chromosome. mRNA levels of glutathione reductase, measured by RT-PCR, increased in response to exposure to 100% ambient oxygen by almost twofold and administration of paraquat by greater than threefold. Exposure of flies to hyperoxia also induced a 60% increase in the activity of glutathione reductase and augmented the concentration of total glutathione by ca. 40% following an initial drop. The present study, besides providing an initial molecular characterization of the glutathione reductase gene in Drosophila, demonstrates its dynamic involvement in response to experimentally induced oxidative stress.

Amino Acid Sequence↗
PubMed1999

Developmental regulation of the translational repressor NAT1 during cardiac development.

The process of translation initiation has been postulated to play an important role in the regulation of cellular growth and proliferation. Here, we report the identification and differential expression of a fundamental translational repressor NAT1, during early postnatal cardiac development. Differential display analysis of RNA obtained from 3-day and 4-week-old rat hearts resulted in the cloning and identification of a 396 bp cDNA fragment (DRCF-6) which corresponded to the 3' terminal portion of NAT1. Northern blot analysis revealed that the mRNA expression of NAT1 was markedly elevated during the first 2 weeks of postnatal life, with an apparent peak level of expression occurring at 1 week. NAT1 mRNA levels then steadily decreased to 4 weeks of age. The NAT1 transcript has previously been shown to be extensively edited by the enzyme APOBEC-1, which deaminates specific cytidine bases to uridine; cytidine deamination at a glutamine codon (CAA) results in the formation of a stop codon (UAA) and consequently, premature termination of translation. Accordingly, Western blot analysis detected the presence of several smaller proteins in addition to the full length NAT1 protein (97 kDa), each exhibiting a distinct pattern of expression during cardiac development. APOBEC-1 editing of NAT1 during cardiac development was further supported by primer extension analysis of cytidine 1699, which was found to be predominantly edited to uridine. Immunohistochemical staining showed that NAT1 is expressed predominantly in atrial and ventricular myocytes, although staining was also detected in vascular smooth muscle cells and in the endocardium. These results suggest that NAT1 may play a role in the postnatal development of the heart and demonstrate that APOBEC-1 editing may possibly be a novel mechanism by which translation is regulated during cardiac development.

Aging↗
PubMed1998

Mutation analysis in familial hypercholesterolemia patients of different ancestries: identification of three novel LDLR gene mutations.

Twelve familial hypercholesterolemia (FH) patients of different ancestries living in South Africa were subjected to mutation analysis of the low-density lipoprotein receptor (LDLR) gene. Nine different mutations were identified in 10 patients. Six of these, including the founder-related mutation C660X identified in two Lebanese patients, have previously been described in other FH patients with compatible genetic backgrounds, and/or in patients originating from countries where admixture is not uncommon. Characterization of an abnormal electrophoresis pattern detected in exon 4 of the LDLR gene by heteroduplex single-strand conformation polymorphism (HEX-SSCP) analysis, revealed a novel G deletion at codon 185 (617delG) which resulted in a downstream stop codon. Two of the new mutations identified resulted in amino acid substitutions and were designated R57C and Q357P.

Apolipoproteins B↗
PubMed1993

Conservation of the segment 4 gene sequence and of a leucine zipper motif in VP4 among five US bluetongue viruses.

Full-length cDNA copies of the segment 4 (M1) genes of US Bluetongue viruses serotype-2, -10, -11, -13, and -17 were selectively amplified using genomic double-stranded RNA segments from purified BTV virions as templates and a modified polymerase chain reaction (Clamp-R). They were then cloned into pUC19 plasmids and both strands of several clones were sequenced. The length of all five segment 4 genes is 1981 nucleotides, which is 30 nucleotides shorter than that of the BTV serotype-10 reported by Y. Yu, A. Fukusho, and P. Roy (Nucleic Acids Res. 15, 7206 (1987)). The 5'- and 3'-noncoding regions of all five segment 4 genes are identical among all serotypes. The plus sense strand of the BTV segment 4 gene, which encodes the VP4 protein, possesses a single long open reading frame with an initiation codon (ATG) at nucleotides 9-11 and a stop codon (TAA) at nucleotides 1941-1943. This open reading frame encodes for a protein of 644 amino acid residues with a predicted molecular weight of about 75 kDa and a pI of +7 to +7.9. A potential leucine zipper motif was detected near the carboxyl terminus of the deduced VP4 amino acid sequence. The phylogenetic analysis using the sequences of these five cognate segment 4 genes is consistent with the results of our previous phylogenetic studies of cognate genome segments 5, 6, 8, 9, and 10. Serotype-10, -11, -13, and -17 are closely related and serotype-2 is the most distantly related among the five US BTV serotypes.

Amino Acid Sequence↗
PubMed1999

Mutations in the C, D, and V open reading frames of human parainfluenza virus type 3 attenuate replication in rodents and primates.

Human parainfluenza virus type 3 (HPIV3) is a single-stranded negative-sense RNA virus belonging to the Respirovirus genus of the Paramyxoviridae family in the order Mononegavirales. The P gene encodes at least four proteins, including the C protein, which is expressed from an open reading frame (ORF) that overlaps the P ORF, and the D protein, which is encoded when the P ORF is fused to the D ORF by transcriptional editing. The P mRNA also contains a third ORF for the V protein, although it is unclear how or whether this ORF is accessed. We have used recombinant DNA technology to recover five mutant viruses that either interrupt or alter the C, D, and V ORFs. In one mutant virus, rC-KO, expression of the C protein was abrogated by changing the start codon from methionine to threonine and introducing two stop codons at amino acid positions 7 and 26 of the C ORF. In a second mutant virus, rF164S, a point mutation was introduced into the C ORF changing amino acid position 164 from phenylalanine (F) to serine (S), which corresponds to the F170S mutation described in the C protein of Sendai virus (Itoh et al., J. Gen. Virol. 78, 3207-3215). rC-KO was significantly attenuated in vitro and in vivo (rodents and primates), whereas rF164S was attenuated only in vivo. Interestingly, the rF164S mutant was more attenuated in the upper than in the lower respiratory tract of hamsters and monkeys. This pattern is the converse of that seen with temperature-sensitive attenuating mutations, and thus inclusion of this novel mutation in a recombinant live-attenuated vaccine candidate might prove useful in reducing residual virulence in the upper respiratory tract. Both rC-KO and rF164S conferred protection against challenge with wild-type HPIV3. In three other viruses, the D and V ORFs were interrupted singly or in combination. Although interruption of the D and V ORFs individually did not affect virus replication in vitro or in vivo, interruption of both together attenuated replication in vivo. These results indicate that the C, D, and V proteins of HPIV3 each has a role in virus replication in vitro, in vivo, or both, and define mutations that might be useful for the development of a vaccine against HPIV3.

Animals↗
PubMed1992

Accumulation of chloroplast psbB RNA requires a nuclear factor in Chlamydomonas reinhardtii.

We have isolated and characterized a nuclear mutant, 222E, in Chlamydomonas reinhardtii, which is defective in photosystem II (PSII). Polypeptide P5, the product of psbB, is not produced in this mutant, leading to a destabilization of other PSII components. The mutant specifically fails to accumulate psbB transcripts and displays an altered transcription pattern downstream of psbB. Pulse-labelling experiments suggest that mRNA stability and/or processing are affected by the alteration of a nuclear gene product in this mutant. We show that the C. reinhardtii psbB gene is co-transcribed with a small open reading frame that is highly conserved in location and amino acid sequence in land plants. The 5' and 3' termini of the psbB transcript have been mapped to 35 bases upstream of the initiation codon and approximately 600 bases downstream of the stop codon. The 3' flanking region contains two potential stem-loops, of which the larger (with an estimated free energy of -46 kcal) is near the 3' terminus of the transcript.

Amino Acid Sequence↗
PubMed1987

The cytochrome oxidase subunit I gene of Tetrahymena: a 57 amino acid NH2-terminal extension and a 108 amino acid insert.

The gene sequence for cytochrome oxidase subunit I (COI) in the ciliate Tetrahymena mitochondrial DNA has been determined and shown to be coded by the same strand as codes the genes (in order) for 14S rRNA, tRNA(trp), tRNA(glu), 21S rRNA, tRNA(leu) and tRNA(met). The predicted protein has 698 amino acids, including an NH2-terminal 57 amino acid extension and a 108 amino acid insert originally found in Paramecium COI. These extension and insert segments are not highly hydrophobic but are relatively rich in lysine, arginine and serine. In analogy with the presequence of nuclear-encoded mitochondrial proteins, they might function as a transmembrane signal. The remaining polypeptide segments show a hydrophobicity characteristic of membrane spanning proteins. TCOI shows a 64% amino acid identity with Paramecium COI but less than a 38% amino acid conservation with human COI. The Tetrahymena mitochondrial code is analogous with the mammalian mitochondrial code; but differs from the Tetrahymena nuclear genetic code; TGA is exclusively translated as tryptophan; ATA is used as an initiation codon probably for methionine, and TAA as a stop codon; the arginine codons (CGN) are not used. The use of the leucine codon TTA in TCOI is contradictory to the codon recognition pattern previously obtained from the isolated tRNA(leu) isoacceptors recognizing only the CUN codons, but consistent with the tRNA(leu) (anticodon UAA) gene encoded in the genome. The reason for this inconsistency has not been resolved.

Amino Acid Sequence↗