Second Position Of Codon Research Articles

Strong Selectional Forces Fine-Tune CpG Content in Genes Involved in Neurological Disorders as Revealed by Codon Usage Patterns.

Neurodegenerative disorders cause irreversible damage to the neurons and adversely affect the quality of life. Protein misfolding and their aggregation in specific parts of the brain, mitochondrial dysfunction, calcium load, proteolytic stress, and oxidative stress are among the causes of neurodegenerative disorders. In addition, altered metabolism has been associated with neurodegeneration as evidenced by reductions in glutamine and alanine in transient global amnesia patients, higher homocysteine-cysteine disulfide, and lower methionine decline in serum urea have been observed in Alzheimer’s disease patients. Neurodegeneration thus appears to be a culmination of altered metabolism. The study’s objective is to analyze various attributes like composition, physical properties of the protein, and factors like selectional and mutational forces, influencing codon usage preferences in a panel of genes involved directly or indirectly in metabolism and contributing to neurodegeneration. Various parameters, including gene composition, dinucleotide analysis, Relative synonymous codon usage (RSCU), Codon adaptation index (CAI), neutrality and parity plots, and different protein indices, were computed and analyzed to determine the codon usage pattern and factors affecting it. The correlation of intrinsic protein properties such as the grand average of hydropathicity index (GRAVY), isoelectric point, hydrophobicity, and acidic, basic, and neutral amino acid content has been found to influence codon usage. In genes up to 800 amino acids long, the GC3 content was highly variable, while GC12 content was relatively constant. An optimum CpG content is present in genes to maintain a high expression level as required for genes involved in metabolism. Also observed was a low codon usage bias with a higher protein expression level. Compositional parameters and nucleotides at the second position of codons played essential roles in explaining the extent of bias. Overall analysis indicated that the dominance of selection pressure and compositional constraints and mutational forces shape codon usage.

GCN sensitive protein translation in yeast.

Levels of protein translation by ribosomes are governed both by features of the translation machinery as well as sequence properties of the mRNAs themselves. We focus here on a striking three-nucleotide periodicity, characterized by overrepresentation of GCN codons and underrepresentation of G at the second position of codons, that is observed in Open Reading Frames (ORFs) of mRNAs. Our examination of mRNA sequences in Saccharomyces cerevisiae revealed that this periodicity is particularly pronounced in the initial codons-the ramp region-of ORFs of genes with high protein expression. It is also found in mRNA sequences immediately following non-standard AUG start sites, located upstream or downstream of the standard annotated start sites of genes. To explore the possible influences of the ramp GCN periodicity on translation efficiency, we tested edited ramps with accentuated or depressed periodicity in two test genes, SKN7 and HMT1. Greater conformance to (GCN)n was found to significantly depress translation, whereas disrupting conformance had neutral or positive effects on translation. Our recent Molecular Dynamics analysis of a subsystem of translocating ribosomes in yeast revealed an interaction surface that H-bonds to the +1 codon that is about to enter the ribosome decoding center A site. The surface, comprised of 16S/18S rRNA C1054 and A1196 (E. coli numbering) and R146 of ribosomal protein Rps3, preferentially interacts with GCN codons, and we hypothesize that modulation of this mRNA-ribosome interaction may underlie GCN-mediated regulation of protein translation. Integration of our expression studies with large-scale reporter studies of ramp sequence variants suggests a model in which the C1054-A1196-R146 (CAR) interaction surface can act as both an accelerator and braking system for ribosome translation.

Codon usage analysis of photolyase encoding genes of cyanobacteria inhabiting diverse habitats.

Nucleotide and amino acid compositions were studied to determine the genomic and structural relationship of photolyase gene in freshwater, marine and hot spring cyanobacteria. Among three habitats, photolyase encoding genes from hot spring cyanobacteria were found to have highest GC content. The genomic GC content was found to influence the codon usage and amino acid variability in photolyases. The third position of codon was found to have more effect on amino acid variability in photolyases than the first and second positions of codon. The variation of amino acids Ala, Asp, Glu, Gly, His, Leu, Pro, Gln, Arg and Val in photolyases of three different habitats was found to be controlled by first position of codon (G1C1). However, second position (G2C2) of codon regulates variation of Ala, Cys, Gly, Pro, Arg, Ser, Thr and Tyr contents in photolyases. Third position (G3C3) of codon controls incorporation of amino acids such as Ala, Phe, Gly, Leu, Gln, Pro, Arg, Ser, Thr and Tyr in photolyases from three habitats. Photolyase encoding genes of hot spring cyanobacteria have 85% codons with G or C at third position, whereas marine and freshwater cyanobacteria showed 82 and 60% codons, respectively, with G or C at third position. Principal component analysis (PCA) showed that GC content has a profound effect in separating the genes along the first major axis according to their RSCU (relative synonymous codon usage) values, and neutrality analysis indicated that mutational pressure has resulted in codon bias in photolyase genes of cyanobacteria.

Hereditary systemic immunoglobulin light-chain amyloidosis

AbstractSeveral members of a family died from renal failure as a result of systemic amyloidosis. Extensive studies to detect previously documented gene mutations associated with amyloidosis failed to identify a causative factor. In search of the genetic basis for this syndrome, amyloid fibrils were isolated from renal tissue of a member of the kin who died while on renal dialysis. Amino acid sequencing of isolated amyloid protein identified sequences compatible with the constant region of the immunoglobulin κ light-chain. Isolation and characterization of κ light-chain protein from serum of an affected member of the kindred revealed mutation in the constant region of κ light-chain, with cysteine replacing serine at amino acid residue 131. This mutation (Ser131Cys) was confirmed by DNA analysis, which identified a single-base change of cytosine to guanine at the second position of codon 131 of the κ light-chain gene (TCT131TGT). DNA analysis of members of the extended family revealed transmission of the Ser131Cys mutation and association with systemic amyloidosis. This amyloid light-chain (AL) amyloidosis, which is a hereditary type of amyloidosis and not the result of a monoclonal plasma cell dyscrasia, may be misdiagnosed and lead to inappropriate chemotherapy.

P53 codon 72 polymorphism is associated with susceptibility to hepatocellular carcinoma in the Turkish population: a case–control study

The tumor suppressor p53 gene plays a crucial role in preventing carcinogenesis through its ability to induce cell cycle arrest and apoptosis following DNA damage and oncogene activation. A guanine (G)/cytosine (C) common single nucleotide polymorphism (SNP) at second position of codon 72 in exon 4 of p53 gene determines a arginine (Arg) to proline (Pro) (Arg72Pro) aminoacidic substitution within the proline-rich domain of p53 protein. Arg72 and Pro72 allele are different from a biochemical and biological point of view and many reports suggest that they can modulate individual cancer susceptibility. To determine the association of the p53 Arg72Pro polymorphism with the risk of hepatocellular carcinoma (HCC) development in a Turkish population, a hospital-based case-control study was designed consisting of 119 subjects with HCC and 119 cancer-free control subjects matched for age, gender, smoking and alcohol status. The genotype frequency of the p53 Arg72Pro polymorphism was determined by using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Our data shows that the Pro/Pro genotype of the p53 Arg72Pro polymorphism is associated with increased risk of HCC development in this Turkish population (OR = 3.20, 95% CI: 1.24-8.22, P = 0.02). Furthermore, according to stratified analysis, a significant association was observed between the homozygote Pro/Pro genotype and HCC risk in the subgroups of male gender (OR = 3.01, 95% CI: 1.14-7.97, P = 0.03) and patients with hepatitis B virus (HBV)-related HCC (OR = 4.04, 95% CI: 1.46-11.15, P = 0.007). Because our results suggest for the first time that the Pro/Pro homozygote of p53 Arg72Pro polymorphism may be a genetic susceptibility factor for HCC (especially in the male gender and HBV-infected patients) in the Turkish population, further independent studies are required to validate our findings in a larger series, as well as in patients of different ethnic origins.

The Post-Transcriptional mRNA Editing Analysis of cox3 Mitochondrial Gene in Fern <i>Asplenium nidus</i> Reveals Important Features

In the mitochondria and chloroplasts of flowering plants (angiosperms), transcripts of protein-coding genes are altered after synthesis so that their final primary nucleotide sequence differs from that of the corresponding DNA sequence. This posttranscriptional mRNA editing consists almost exclusively of C-to-U substitutions (direct) and less frequently of U-to-C substitution (reverse). Editing occurs predominantly within coding regions, mostly at isolated C residues, and usually at first or second positions of codons, thereby almost always changing the amino acid from that specified by the unedited codon. Editing may also create initiation and termination codons. The effect of C-to-U RNA editing in plants is to make proteins encoded by plant organelles more similar in sequence to their non plant homologs, then specific C-to- U editing events are essential for the production of functional plant mitochondrial proteins. Our attention has been devoted to the study of the mRNA editing in cox3 mitochondrial gene of fern Asplenium nidus. This study reveals the extreme importance of both C-to-U and U-to-C substitutions for protein expression.

A novel F1552/C1552 point mutation in the Aedes aegypti voltage-gated sodium channel gene associated with permethrin resistance

Sodium channel mutations were investigated through nucleotide sequencing of three cDNA fragments amplified from permethrin resistant and susceptible Aedes aegypti from northern Thailand. There was a novel nucleotide substitution (T → G) at the second position of codon 1552 resulting in the replacement of Phenylalanine by Cysteine in segment 6 domain III. This amino acid was indicated by another study to involve an aromatic–aromatic contact between the sodium channel protein and the first aromatic ring of the pyrethroid alcohol moiety. Reciprocal crosses between the homozygous parental susceptible and resistant strains indicated that resistance was autosomal and incompletely recessive, and highly associated with the homozygous mutation. The bioassay of the F 2 progeny, formed by backcrossing the F 1 with the resistant parental strain, did not show a clear plateau curve across the range of doses, suggesting that resistance to permethrin was controlled by more than one gene locus. Other possible resistance mechanisms involved are discussed.

The mitochondrial genome structure of the clouded leopard (Neofelis nebulosa)

The complete 16 844 bp mitochondrial genome of Neofelis nebulosa has been sequenced and compared with the complete mitochondrial genomes of Felis catus and the Acinonyx jubatus. The base composition of the mitochondrial genome of N. nebulosa is as follows: A, 5343 bp (31.7%); C, 4441 bp (26.4%); G, 2491 bp (14.8%); T, 4569 bp (27.1%). The genome complement and the gene order of this mitochondrial genome was found to be typical of those reported for other mammals. Several unusual features of this genome, however, were found. First, in protein-coding regions, AT bias in the genome was not prevalent in the third position of codons, as it is in most other mammals, but was found in the second position of codons. Second, in tRNA regions, tRNASer (AGY), which lacked the "DHU" arm, could not be folded into the typical cloverleaf-shaped structure. Third, in the control region, no repetitive sequences (RS)-2 were found. However, RS-2 repetitive motifs usually occurr in the control regions of most great cats. In addition, 4 variable sites were found in CSB-3 of the control region. Fourth, AT content in the control region of the mtDNA from the clouded leopard was lower than it is in other regions.

Novel mutation (Asp158Val) in H1 domain of keratin 5 gene in a Japanese patient with Köbner‐type epidermolysis bullosa simplex

We describe a 19-year-old Japanese male with Köbner-type epidermolysis bullosa simplex (EBS-KB) with a novel keratin gene mutation. The patient developed blisters on the feet, palms, elbows and knees soon after birth. His father is similarly affected with blistering, but his mother and younger brother are not affected. Histological examination revealed that the primary separation in the blister occurred within the basal cell layer. Sequence analysis demonstrated an A-to-T transition at the second position of codon 158 in the keratin 5 (K5) gene. The amino acid at codon 158 was deduced to have changed from asparagine to valine. We identified a novel mutation (Asp158Val) in the H1 domain of the K5 gene in this Japanese patient with EBS-KB. This is the first gene mutation report of EBS-KB in the H1 domain of the K5 gene.

Multiplex Pyrosequencing of Two Polymorphisms in DNA Repair Gene XRCC1

DNA repair enzymes play a pivotal role in platinum-based chemotherapy. Within the gene encoding for the base excision repair enzyme XRCC1, several nonsynonymous polymorphisms have been identified. It has been shown that the Arg399Gln single-nucleotide polymorphism results in a polymorphic enzyme that is less capable of initiating DNA repair. We developed a multiplex pyrosequence assay to simultaneously detect two nonsynonymous polymorphisms within the XRCC1 gene. Both of these polymorphisms resulted in amino acid changes: G/A in codon 399 changes Arg into Gln, and deletion of A in the second position of codon 576 results in a stopcodon. We established the frequency of these mutations in 270 patients suffering from colorectal cancer. Allele frequencies of G in second position of codon 399 and A in the second position codon 576 are 61.1 and 99.6%, respectively, in these patients. This fast and reliable method allows for simultaneous detection of the infrequent mutant C or CT alleles instead of the A deletion at codon 576. The method may be used in pharmacogenetic studies of platinum-based chemotherapy.

Genetic Polymorphism of Interferon-γ, Interferon-γ Receptor, and Interferon Regulatory Factor-1 Genes in Patients with Hepatitis B Virus Infection

The natural history of hepatitis B virus (HBV) infection is probably related to host immune factors. Interferon-gamma (IFN-gamma) plays significant roles in immune defense. This study was undertaken to investigate the association between HBV infection and single nucleotide polymorphisms (SNPs) of IFN-gamma, IFN-gamma receptor (IFNGR)-1 and 2, and interferon regulatory factor (IRF)-1 genes. Between March 2002 and December 2002, 614 Korean patients were enrolled in two different groups: an HBV clearance group (n = 201), who were hepatitis B surface antigen (HBsAg) negative with antibodies to HBsAg and hepatitis B core antigen, and an HBV persistence group (n = 413), who were repeatedly HBsAg positive. We assessed polymorphisms in the IFN-gamma gene at position +874, in the IFNGR-1 gene at positions -56 and +95, in the IFNGR-2 gene at the second position of codon 64 (Gln64Arg), and in the IRF-1 gene promoter (-410, -388), and the genotype distributions of the HBV clearance and persistence groups were compared. On the basis of unconditional logistic regression analysis with adjustment for age and sex, no statistically significant association with susceptibility to persistent HBV infection was observed with the IFN-gamma, IFNGR-1 and 2, and IRF-1 gene polymorphisms under the codominant, dominant, and recessive models.

His239Arg SNP of HRAD9 is associated with lung adenocarcinoma

It was previously reported that a functional human (h) Rad9 protein accumulated in the nuclei of non-small cell lung carcinoma (NSCLC) cells. Those experiments, however, did not examine whether the hRad9 gene was mutated in those cells. The sequence of the HRAD9 gene in NSCLC cells was investigated. The sequence of the HRAD9 was examined in tumor and peripheral normal lung tissues obtained from 50 lung adenocarcinoma patients during surgery. The expression of its mRNA using reverse transcription polymerase chain reaction (RT-PCR) was also examined. No sequence alterations were detected in the HRAD9 gene, which was found to be normally transcribed in surgically resected lung carcinoma cells. However, in eight (16.0%) cases a single nucleotide polymorphism (SNP) was observed at the second position of codon 239 (His/Arg heterozygous variant) of the gene. This frequency was significantly higher than that found in the normal population. Whereas the capacity to produce a functional hRad9 protein was intact in lung adenocarcinoma cells, a nonsynonymous SNP of HRAD9 was detected that might be associated with the development of lung adenocarcinoma.

DNA Sequence of Hemoglobin Chico.

We were asked to investigate a 54 year old male who was undergoing evaluation for sleep apnea. The patient had a low resting O2 saturation at 91%, but no signs of cardiac or pulmonary disease as indicated by a normal echocardiogram, normal spirometry, and normal high resolution CT of the chest. Arterial blood gases revealed a pO2 of 79 mm Hg on room air, but pCO2 was normal. The patient's hemoglobin concentration was 13.7 g/dL. The possibility of a mutant hemoglobin was raised and HPLC revealed HbA2 = 2.6%, HbA = 51.4% and an unidentified mutant hemoglobin of 46%. Oxygen dissociation testing demonstrated a P50 of 39 mm Hg (normal 24–32) consistent with a low affinity hemoglobin. The isopropanol test showed that the hemoglobin variant was unstable. Trypsin digestion of the abnormal hemoglobin resulted in a smaller than normal HPLC peak for the β9 segment of β-globin, which is composed of amino acids 67 through 82, and the appearance of a new peak at 193 min. This peak did not match any of the known mutant hemoglobins contained in our reference bank. We PCR amplified and sequenced the β-globin gene. The patient was heterozygous for an A to C mutation in the second position of codon 66. This mutation changed the lysine to threonine and revealed that the hemoglobin variant was Hb Chico. This result was consistent with the loss of ~50% of the β9 peak in the trypsin digest due to loss of a cleavage site after amino acid 66, and the appearance of a new peak which was a hybrid segment of β8 and β9. This represents the first DNA sequence of hemoglobin Chico a mildly unstable hemoglobin variant with decreased oxygen affinity.

The First Indian Mutation of NADH-Cytochrome b5 Reductase as a Cause for Type 1 Methemoglobinemia.

Congenital methemoglobinemia caused by cytochrome b5 reductase(b5R) deficiency is sporadic worldwide, although clusters of this disorder have been identified in certain ethnic groups. Several case reports of congenital methemoglobinemia have been reported from India, most with cytochrome b5 reductase deficiency. However, as of yet, no cytochrome b5 reductase Indian mutation has been identified. Here we describe the first molecular basis of b5R deficiency to cause methemoglobinemia in India. The propositus is a 22 year old Indian male who was born from a consanguineous union in Southern India. He was cyanotic since birth. He had no neurologic deficits and no known exposures to drugs or toxins. Congenital methemoglobinemia was suspected after an extensive cardiopulmonary workup was unrevealing. He had an elevated methemoglobin level of 16%. Hemoglobin M was not present. Spectrophotometric assay of b5R activity in erythrocytes was 4% of normal mean, while his father had normal enzyme activity and his mother and sister had 35 and 45% of normal, respectively. b5R activity in the propositus' platelets and granulocytes was normal, consistent with a diagnosis of type 1 methemoglobinemia. Genomic DNA was PCR amplified and sequenced. A mutation was found in exon 8 in the second position of codon 217 which is a T to C transversion at position 25985 in the gene which changes the amino acid at that position from leucine to proline(L217P). The propositus was homozygous for the mutation, while both parents and sister were heterozygous for this mutation, confirming the autosomal recessive pattern of inheritance of type 1 methemoglobinemia. b5R mRNA levels were elevated in all family members, a finding not previously reported with any b5R mutation. We also show that measurement of enzyme activity is not reliable for detection of heterozygosity of b5R deficiency as his father had normal enzyme activity. The L217P mutation, located in the NADH binding domain, lies in a region of the sequence that is highly conserved throughout evolution from yeast to man, suggesting its essential importance in enzyme function. We examined the effect of this mutation by replacing the wild-type residue with the mutant one in the human b5R structure(PDB:1UMK) using the program Insight II. As residue 217 lies at the base of alpha helix Nα3, the L217P mutation causes steric hindrance with contacts between the proline side-chain and the main chain before energy minimization. Following energy minimization, no major changes in main-chain path were observed. However, contact alterations with residues within 3.0A radius of residue 217 were recorded. Since residue 217 is located within a hydrophobic pocket we also examined the effect of the mutation on solvent accessibility. Our model predicts that glutamate 220, a polar amino acid, is buried deeper in the hydrophobic pocket in the mutant protein. Assuming our model is accurate, the mutation is predicted to result in enzyme instability, thus explaining the type 1 phenotype of the propositus. Biochemical studies are underway to test the validity of our model. It remains to be seen if this disorder is endemic in India as it is in other ethnic groups. [Display omitted]

Mitochondrial Cytochrome b mRNA Editing in Dinoflagellates: Possible Ecological and Evolutionary Associations?

To verify the hypothesis that mt mRNA editing is widespread in dinoflagellates, we analyzed cytochrome b (cob) mRNA editing for six species representing distinct ecotypes and taxonomic classes of Dinophyceae. Editing is detected in all, which is similar to the three other species studied previously in that edited sites appear to aggregate in four clusters and occur predominantly at first and second positions of codons (93%), overwhelmingly involving A --> G, U --> C, or C --> U substitutions with a smaller number of G --> C, G --> A changes. Comparative analyses on editing characteristics reveal interesting trends related to phylogenetic relatedness and ecological features. Editing density (percentage of nucleotide that is affected by editing) increases from early to derived lineages. Higher editing densities also map to red tide-forming lineages. Furthermore, similarity of location of edited codons (LOE) and the type of nucleotide changes (TOE) in different lineages mirror the taxonomic affinity of the lineages. Phylogenetic trees constructed from LOE and TOE resemble those inferred from cob sequences. The results bolster our earlier hypothesis that cob editing is widespread in dinoflagellates and suggest that density, location, and type of editing may bear yet-to-be-defined evolutionary and ecological significance.

Dual roles of glycosyl torsion angle conformation and stereochemical configuration in butadiene oxide-derived N1 beta-hydroxyalkyl deoxyinosine adducts: a structural perspective.

The solution structure of the N1-[1-hydroxy-3-buten-2(R)-yl]-2'-deoxyinosine adduct arising from the alkylation of adenine N1 by butadiene epoxide (BDO), followed by deamination to deoxyinosine, was determined in the oligodeoxynucleotide 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTTGTCCG)-3'. This oligodeoxynucleotide contained the BDO adduct at the second position of codon 61 of the human N-ras protooncogene (underlined) and was named the ras61 R-N1-BDO-(61,2) adduct. 1H NMR revealed a weak C5 H1' to X6 H8 nuclear Overhauser effects (NOE), followed by an intense X6 H8 to X6 H1' NOE. Simultaneously, the X6 H8 to X6 H3' NOE was weak. The resonances arising from the T16 and T17 imino protons were not observed. 1H NOEs between the butadiene moiety and the DNA positioned the adduct in the major groove. Structural refinement based upon a total of 394 NOE-derived distance restraints and 151 torsion angle restraints yielded a structure in which the modified deoxyinosine was in the syn conformation about the glycosyl bond, with a glycosyl bond angle of 83 degrees , and T17, the complementary nucleotide, was stacked into the helix but not hydrogen bonded with the adducted inosine. The refined structure provides a plausible hypothesis as to why these N1 deoxyinosine adducts strongly code for the incorporation of dCTP during trans lesion DNA replication, irrespective of stereochemistry, both in Escherichia coli [Rodriguez, D. A., Kowalczyk, A., Ward, J. B. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2001) Environ. Mol. Mutagen. 38, 292-296] and in mammalian cells [Kanuri, M., Nechev, L. N., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2002) Chem. Res. Toxicol. 15, 1572-1580]. Rotation of the N1 deoxyinosine adduct into the syn conformation may facilitate incorporation of dCTP via Hoogsteen type templating with deoxyinosine, generating A to G mutations. However, conformational differences between the R- and the S-N1-BDO-(61,2) adducts, involving the positioning of the butenyl moiety in the major groove of DNA, suggest that adduct stereochemistry plays a secondary role in modulating the biological response to these adducts.

Estimating Divergence Times in Phylogenetic Trees Without a Molecular Clock

were nonstationary at first positions for the ingroup at P = 0.01 using the chi-square test in PAUP*. The chi-square test in PAUP∗ appears to do a good job of identifying the worst offenders and although crude, is effective for a large data set when analyzing individual genes. We certainly anticipate that as more refined tests of stationarity are implemented (e.g., Foster, 2004), discrimination of genes that deviate from the stationary condition will improve. We note finally that although deviations from stationarity were detected at third positions, we are not suggesting that all misleading base compositional signal is at third positions. Since these nucleotides are linked as codons, we might expect that strong deviations at third positions would influence first and second positions of codons. Broadly speaking, accurate phylogenetic trees can be recovered from correctly aligned sequences when the inference model is consistent with the process that gave rise to the data. When processes are stationary over lineages and time, relatively straightforward models can be designed to yield accurate inferences, even from short sequences (Steel and Penny, 2000). When processes differ across or within lineages, models must explicitly accommodate the nonstationarity involved. This is generally not straightforward, and even if it could be done, would require many more parameters and associated error terms (but see Foster, 2004). As such, at a given data set size, stationary sequences will prove to be more effective for recovering phylogeny. Stationary sequences will be less prone to the grouping of taxa with convergent base compositions. Of course, when taxa share an atypical base composition in a gene sequence because of shared history, nonstationary sequences may outperform stationary sequences in recovering that branch when using models that assume stationarity. Such instances are cases of obtaining the right answer for the wrong reason (e.g. Swofford et al. 2001) and are a poor argument for use. The criterion of stationarity should prove useful in selecting genes for phylogenetic analysis from completely sequenced genomes, and to the extent that genes

Hereditary Lattice Corneal Dystrophy Is Associated with Corneal Amyloid Deposits Enclosing C-Terminal Fragments of Keratoepithelin

To investigate the molecular basis of hereditary lattice corneal dystrophy (LCD) type IIIA associated with corneal amyloid deposits afflicting several members of a four-generation family. Histologic, immunohistochemical and biochemical studies were performed on corneal tissue samples obtained after perforating keratoplasty. DNA was extracted from peripheral blood leukocytes. All exons of the keratoepithelin-encoding TGFBI gene were amplified and sequenced. The presence of a mutation was confirmed by digestion of the isolated PCR product with the restriction enzyme AlwNI. The cornea of the index patient (II-1) contained large patchy deposits of amyloid, which were immunoreactive for the C terminus of keratoepithelin. Western blot analysis of the polypeptide chains extracted from the amyloid deposits of paraffin-embedded tissue revealed that these represented mainly fragments of the full-length protein. The smallest fragments were 6.5 and 6.9 kDa. DNA analyses of the TGFBI gene revealed a heterozygous T-->C transition at the second position of codon 540 in exon 12, indicating that replacement of phenylalanine by serine (Phe540Ser) leads to dominant disease. The mutation creates a new restriction site for the enzyme AlwNI. Five of the examined family members carried this mutation. Three of them (aged >/=41 years) had the disease, two family members (aged <20 years) do not yet show any clinical symptoms. An additional inconsequential single-nucleotide polymorphism (T1667C) was found at the third position of the same codon (Phe540Phe) in three unaffected family members. This is the first report of a single-nucleotide mutation at codon 540 of TGFBI leading to LCD, and the first to demonstrate that the amyloid deposits in LCD contain proteolytic fragments of keratoepithelin.

Structure of an Oligodeoxynucleotide Containing a Butadiene Oxide-Derived N1 Beta-Hydroxyalkyl Deoxyinosine Adduct in the Human N-ras Codon 61 Sequence

The solution structure of the N1-(1-hydroxy-3-buten-2(S)-yl)-2'-deoxyinosine adduct arising from the alkylation of adenine N1 by butadiene epoxide (BDO), followed by deamination to deoxyinosine, was determined, in the oligodeoxynucleotide d(CGGACXAGAAG).d(CTTCTCGTCCG). This oligodeoxynucleotide contained the BDO adduct at the second position of codon 61 of the human N-ras protooncogene, and was named the ras61 S-N1-BDO-(61,2) adduct. (1)H NMR revealed a weak C(5) H1' to X(6) H8 NOE, followed by an intense X(6) H8 to X(6) H1' NOE. Simultaneously, the X(6) H8 to X(6) H3' NOE was weak. The resonance arising from the T(17) imino proton was not observed. (1)H NOEs between the butadiene moiety and the DNA positioned the adduct in the major groove. Structural refinement based upon a total of 364 NOE-derived distance restraints yielded a structure in which the modified deoxyinosine was in the high syn conformation about the glycosyl bond, and T(17), the complementary nucleotide, was stacked into the helix, but not hydrogen bonded with the adducted inosine. The refined structure provided a plausible hypothesis as to why this N1 deoxyinosine adduct strongly coded for the incorporation of dCTP during trans lesion DNA replication, both in Escherichia coli [Rodriguez, D. A., Kowalczyk, A., Ward, J. B. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2001) Environ. Mol. Mutagen. 38, 292-296], and in mammalian cells [Kanuri, M., Nechev, L. N., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2002) Chem. Res. Toxicol. 15, 1572-1580]. Rotation of the N1 deoxyinosine adduct into the high syn conformation may facilitate incorporation of dCTP via Hoogsteen-type templating with deoxyinosine, thus generating A-to-G mutations.

Mispairing of a Site Specific Major Groove (2S,3S)-N6-(2,3,4-Trihydroxybutyl)-2‘-deoxyadenosyl DNA Adduct of Butadiene Diol Epoxide with Deoxyguanosine: Formation of a dA(anti)·dG(anti) Pairing Interaction

The (2S,3S)-N6-(2,3,4-trihydroxybutyl)-2'-deoxyadenosyl (BDT) adduct arising from alkylation of adenine N6 by butadiene diol epoxide (BDE) was placed opposite a mismatched deoxyguanosine nucleotide in the complementary strand of the oligodeoxynucleotide 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTGGTCCG)-3'. This oligodeoxynucleotide contains codon 61 (underlined) of the human N-ras protooncogene. The BDT adduct was at the second position of codon 61, and this was named the ras61 S,S-BDT-(61,2) A.G adduct. NMR spectroscopy revealed the presence of two conformations of the adducted mismatched duplex. In the major conformation, the mismatched base pair X6.G17 was oriented in a "face-to-face" orientation, in which both the modified nucleotide X6 and its complement G17 were intrahelical and in the anti conformation about the glycosyl bond. Hydrogen bonding was suggested between X6 N1 and G17 N1H and between X6 N6H and G17 O6. The presence of the BDT moiety allowed formation of a stable A.G mismatch pair. The identity of the minor conformation could not be determined. If not repaired, the resulting mismatch pair would generate A-->C mutations, which have been associated with this adenine N6 BDT adduct [Carmical, J. R., Nechev, L. N., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2000) Env. Mol. Mutagen. 35, 48-56].