First Position Of Codon Research Articles

Free energy landscape of RNA binding dynamics in start codon recognition by eukaryotic ribosomal pre-initiation complex.

Specific interaction between the start codon, 5'-AUG-3', and the anticodon, 5'-CAU-3', ensures accurate initiation of translation. Recent studies show that several near-cognate start codons (e.g. GUG and CUG) can play a role in initiating translation in eukaryotes. However, the mechanism allowing initiation through mismatched base-pairs at the ribosomal decoding site is still unclear at an atomic level. In this work, we propose an extended simulation-based method to evaluate free energy profiles, through computing the distance between each base-pair of the triplet interactions involved in recognition of start codons in eukaryotic translation pre-initiation complex. Our method provides not only the free energy penalty for mismatched start codons relative to the AUG start codon, but also the preferred pathways of transitions between bound and unbound states, which has not been described by previous studies. To verify the method, the binding dynamics of cognate (AUG) and near-cognate start codons (CUG and GUG) were simulated. Evaluated free energy profiles agree with experimentally observed changes in initiation frequencies from respective codons. This work proposes for the first time how a G:U mismatch at the first position of codon (GUG)-anticodon base-pairs destabilizes the accommodation in the initiating eukaryotic ribosome and how initiation at a CUG codon is nearly as strong as, or sometimes stronger than, that at a GUG codon. Our method is expected to be applied to study the affinity changes for various mismatched base-pairs.

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.

Hematological Characterizations and Molecular Diagnostic Aspects of Hb Wiangpapao [α44(CE2)Pro→Ser (α1), CCG>TCG; HBA1: c.133C>T], a New α-Globin Variant Found in a Pregnant Thai Woman

We report the hematological parameters and provide a rapid molecular analysis method for detection of Hb Wiangpapao [α44(CE2)Pro→Ser, CCG>TCG; HBA1: c.133C>T], a new α-globin variant found in a pregnant Thai woman. Her red cell indices were measured by an automated blood counter. The results were: red blood cell (RBC) count 4.03 × 1012/L, Hb 13.1 (g/dL), packed cell volume (PCV) 0.39 L/L, mean corpuscular volume (MCV) 97.0 fL, mean corpuscular hemoglobin (Hb) (MCH) 32.5 pg, mean corpuscular Hb concentration (MCHC) 33.4 g/dL, and RBC distribution width (RDW) 9.4%. The Hb typing by high performance liquid chromatography (HPLC) showed 13.6% abnormal Hb at a retention time of 2.20 min. that was difficult to distinguish from Hb A. On the capillary electrophoresis (CE) electropherogram, this hemoglobinopathy peak did not separate from the Hb A peak. DNA sequencing showed a C>T transition at the first position of codon 44 (CCG>TCG) of the α1-globin gene that led to a substitution of proline for serine. This mutation has not been recorded in the public databases. Therefore, we named it Hb Wiangpapao as it was first discovered in the Wiangpapao District, Chiang Rai, Thailand. The multiplex allele-specific polymerase chain reaction (ASPCR) for detection of Hb Wiangpapao was developed and revealed a 510 bp specifically amplified fragment. The better understanding of hematological characterizations and the newly developed multiplex ASPCR for diagnosis of Hb Wiangpapao are useful for genetic counseling and family education.

A new lysozyme tyr54asn mutation causing amyloidosis in a family of Swedish ancestry with gastrointestinal symptoms

Familial amyloidoses are a group of inherited disorders that cause deposition of misfolded amyloidogenic proteins in various tissues, resulting in organ dysfunction. Point mutations in the coding region of seven different genes are known to cause clinically significant systemic amyloid disease. We describe a new mutation in exon 2 of the lysozyme gene associated with amyloidosis (ALys) in a 61-year-old woman with a 7-year history of non-bloody, watery diarrhea, and weight loss. Biopsies of the duodenum and stomach were positive for amyloid deposits in the lamina propria and blood vessels. Direct DNA sequencing of the lysozyme gene revealed a single base nucleotide transversion from T to A at the first position of codon 54, resulting in replacement of Tyr by Asn in the mature lysozyme protein (pTyr54Asn). Immunoblot analysis of amyloid fibrils extracted from a fat tissue sample confirmed lysozyme as the amyloid protein. Clinically, the phenotype associated with this lysozyme mutation featured chronic abdominal pain, diarrhea, weight loss, malabsorption, and sicca syndrome. There was no associated nephropathy as has been reported for other ALys mutations. We describe a new mutant lysozyme that presents with abdominal discomfort, diarrhea, weight loss, and sicca syndrome.

Genomic Sequencing of Key Genes in Mouse Pancreatic Cancer Cells

Pancreatic cancer is a multiple genetic disorder with many mutations identified during the progression. Two mouse pancreatic cancer cell lines were established which showed different phenotype in vivo: a non-metastatic cell line, Panc02, and a highly metastatic cell line, Panc02-H7, a derivative of Panc02. In order to investigate whether the genetic mutations of key genes in pancreatic cancer such as KRAS, TP53 (p53), CDKN2A (p16), SMAD4, ZIP4, and PDX-1 contribute to the phenotypic difference of these two mouse pancreatic cancer cells, we sequenced the exonic regions of these key genes in both cell lines and in the normal syngeneic mouse pancreas and compared them with the reference mouse genome sequence. The exons of KRAS, SMAD4, CDKN2A (p16), TP53 (p53), ZIP4, and PDX-1 genes were amplified and the genotype of these genes was determined by Sanger sequencing. The sequences were analyzed with Sequencher software. A mutation in SMAD4 was identified in both cell lines. This homozygote G to T mutation in the first position of codon 174 (GAA) generated a stop codon resulting in the translation of a truncated protein. Further functional analysis indicates that different TGF-β/SMAD signaling pathways were involved in those two mouse cell lines, which may explain the phonotypic difference between the two cells. A single nucleotide polymorphism (SNP) in KRAS gene (TAT to TAC at codon 32) was also identified in the normal pancreas DNA of the syngenic mouse and in both derived tumoral Panc02 and Panc02-H7 cells. No mutation or SNP was found in CDKN2A (p16), TP53 (p53), ZIP4, and PDX-1 genes in these two cell lines. The absence of mutations in genes such as KRAS, TP53, and CDKN2A, which are considered as key genes in the development of human pancreatic cancer suggests that SMAD4 might play a central and decisive role in mouse pancreatic cancer. These results also suggest that other mechanisms are involved in the substantial phenotypic difference between these two mouse pancreatic cancer cell lines. Further studies are warranted to elucidate the molecular pathways that lead to the aggressive metastatic potential of Panc02-H7.

RASSF1A Ala133Ser polymorphism is associated with increased susceptibility to hepatocellular carcinoma in a Turkish population

AimThe tumor suppressor gene Ras association domain family 1 isoform A (RASSF1A) regulates cell cycle regulation, apoptosis and microtubule stability and is inactivated by promoter hypermethylation at a high frequency in hepatocellular carcinoma (HCC). A guanine (G)/thymine (T) common single nucleotide polymorphism (SNP) at first position of codon 133 in RASSF1A gene determines an alanine (Ala) to serine (Ser) (Ala133Ser) amino acidic substitution which may alter cancer risk by influencing the function of RASSF1A protein. MethodsTo determine the association of the RASSF1A Ala133Ser polymorphism with the risk of HCC development in a Turkish population, a hospital-based case–control study was designed consisting of 236 subjects with HCC and 236 cancer-free control subjects matched for age, gender, smoking and alcohol status. The genotype frequency of the RASSF1A Ala133Ser polymorphism was determined by using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. ResultsAllele and genotype associations of RASSF1A Ala133Ser polymorphism with HCC susceptibility were observed in comparisons between the patient and control samples (P<0.001). Risk of HCC development in this Turkish population was significantly increased in carriers of the Ser133 variant allele of Ala133Ser polymorphism (Ala/Ser and Ser/Ser genotypes) when compared with homozygote Ala/Ala genotype (OR=5.47, 95% CI=3.63–8.25, P=0.001). ConclusionBecause our results suggest for the first time that the Ser133 allele of RASSF1A Ala133Ser polymorphism may be a genetic susceptibility factor for HCC 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 Glu727 allele of thyroid stimulating hormone receptor gene is associated with osteoporosis

Background:Published data indicate that thyroid stimulating hormone receptor (TSHR) activities are associated with osteoporosis in some patients.Aim:This study aimed to elucidate whether a given polymorphism of the TSHR gene is associated with osteoporosis.Materials and Methods:One hundred and fifty subjects with osteoporosis were recruited in this study. The diagnosis of osteoporosis was performed with quantitative ultrasound system. The TSHR gene polymorphism was examined by polymerase chain reaction–restriction fragment length polymorphism.Results:The results showed a nucleotide substitution in the first position of codon 36 of the TSHR gene. The nucleotide substitution was from G to C, leading to a 36D → 36H change (D36H) in the predicted amino acid sequence of the receptor. The change did not show significance between healthy subjects and patients with osteoporosis (P > 0.05). On the other hand, we identified another single nucleotide polymorphism that is a C-to-G substitution at codon 727 (GAC to GAG); its frequency was significantly higher in patients with osteoporosis than that in healthy subjects. Using logistic regression analysis, significant correlation was revealed between the genotype D727E and the serum levels of TSH, or the quantitative ultrasound value of the calcaneal bone.Conclusions:The present study suggests that the genotype D727E of the TSHR, but not the genotype D36H, may be a genetic risk factor for osteoporosis.

The significance of Exonuclease 1 K589E polymorphism on hepatocellular carcinoma susceptibility in the Turkish population: a case–control study

Exonuclease 1 (Exo 1) is an important nuclease involved in mismatch repair system that contributes to maintain genomic stability, to modulate DNA recombination, and to mediate cell cycle arrest. A guanine (G)/adenine (A) common single nucleotide polymorphism at first position of codon 589 in Exo 1 gene determines a glutamic acid (Glu, E) to lysine (Lys, K) (K589E) aminoacidic substitution which may alter cancer risk by influencing the activity of Exo 1 protein. Exo 1 K589E polymorphism has been studied in various cancers, but its association with hepatocellular carcinoma (HCC) has yet to be investigated. To determine the association of the Exo 1 K589E polymorphism with the risk of HCC development in a Turkish population, a hospital-based case-control study was designed consisting of 224 subjects with HCC and 224 cancer-free control subjects matched for age, gender, smoking and alcohol status. The genotype frequency of the Exo 1 K589E polymorphism was determined by using a polymerase chain reaction-restriction fragment length polymorphism assay. Our data shows that the Lys/Lys genotype of the Exo 1 K589E polymorphism is associated with increased risk of HCC development in this Turkish population [odds ratio (OR)=2.15, 95% confidence interval (CI): 1.13-4.09, P=0.02]. Furthermore, according to stratified analysis, a significant association was observed between the homozygote Lys/Lys genotype and HCC risk in the subgroups of male gender (OR=2.67, 95% CI: 1.27-5.61, P=0.009) and patients with non-viral-related HCC (OR=3.14, 95% CI: 1.09-8.99, P=0.03). Because our results suggest for the first time that the Lys/Lys homozygote genotype of Exo 1 K589E polymorphism may be a genetic susceptibility factor for HCC 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.

A novel presenilin 1 mutation (L282F) in familial Alzheimer’s disease

Sirs, Mutations in the genes of b-amyloid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2) have been identified as causative in familial Alzheimer’s disease (FAD) [9]. Mutations in PSEN1 are most common in FAD, and 168 pathogenic PSEN1 mutations have been reported to date [3]. We report a novel mutation of PSEN1 (L282F) in a patient with FAD. A 57-year-old woman consulted our hospital because of progressive memory loss. She had noticed forgetfulness since age 53 and had been treated with donepezil at a local clinic since age 55. She presented with impairment of recent memory and orientation for date and scored 24/30 on the mini-mental state examination without any other neurological deficits. Findings of routine blood tests were normal. Serum apolipoprotein E phenotype was E3/E4. Mild medial temporal atrophy was demonstrated on brain magnetic resonance imaging. Fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET) revealed glucose hypometabolism in the bilateral parietal cortices and posterior cingulate gyri. Her family history suggested FAD, but we could not determine the hereditary pattern of FAD (Fig. 1). After written consent was obtained from the patient and her family, all exons of APP, PSEN1 and PSEN2 were analyzed by direct cycle sequencing as described previously [5, 10]. The analysis of PSEN1 demonstrated a C ? T transversion at the first position of codon 282 in exon 8 (genomic accession number: g. 50029C[T), which predicted a L282F substitution (Fig. 2). There were no other mutations detected in the analyzed genes. We also searched for this mutation in 192 Japanese patients with Alzheimer’s disease (AD) or other forms of dementia, and none demonstrated this PSEN1 mutation. We diagnosed this patient as having probable AD according to the NINCDS-ADRDA criteria [6]. The pattern of brain hypometabolism on FDG-PET supported the diagnosis [2, 7]. Although this L282F mutation has not been described previously, two other mutations at the same

A mutation in the voltage‐gated sodium channel gene associated with pyrethroid resistance in Latin American Aedes aegypti

Pyrethroids are commonly used as mosquito adulticides and evolution of resistance to these compounds is a major threat to public health. 'Knockdown resistance' to pyrethroids (kdr) is frequently caused by nonsynonymous mutations in the voltage-gated sodium channel transmembrane protein (para) that reduce pyrethroid binding. Early detection of kdr is critical to the development of resistance management strategies in mosquitoes including Aedes aegypti, the most prevalent vector of dengue and yellow fever viruses. Brengues et al. described seven novel mutations in hydrophobic segment 6 of domain II of para in Ae. aegypti. Assays on larvae from strains bearing these mutations indicated reduced nerve sensitivity to permethrin inhibition. Two of these occurred in codons Iso1011 and Val1016 in exons 20 and 21 respectively. A transition in the third position of Iso1011 encoded a Met1011 replacement and a transversion in the second position of Val1016 encoded a Gly1016 replacement. We have screened this same region in 1318 mosquitoes in 32 additional strains; 30 from throughout Latin America. While the Gly1016 allele was never detected in Latin America, we found two new mutations in these same codons. A transition in the first position of codon 1011 encodes a Val replacement while a transition in the first position of codon 1016 encodes an Iso replacement. We developed PCR assays for these four mutations that can be read either on an agarose gel or as a melting curve. Selection experiments, one with deltamethrin on a field strain from Santiago de Cuba and another with permethrin on a strain from Isla Mujeres, Mexico rapidly increased the frequency of the Iso1016 allele. Bioassays of F(3) offspring arising from permethrin susceptible Val1016 homozygous parents and permethrin resistant Iso1016 homozygous parents show that Iso1016 segregates as a recessive allele in conferring kdr. Analysis of segregation between alleles at the 1011 and 1016 codons in the F(3) showed a high rate of recombination even though the two codons are only separated by a ~250 bp intron. The tools and information presented provide a means for early detection and characterization of kdr that is critical to the development of strategies for resistance management.

Hipoplasia adrenal, hipogonadismo y talla baja por una nueva mutación del gen DAX-1 (pGly168fsX17)

Various genes play a role in the morphogenesis of the adrenal cortex, among them the DAX-1 gene. We report an 18-year-old man who showed complete adrenal failure in the neonatal period, hypogonadotropic hypogonadism and pathological short stature associated with a mutation of the DAX-1 gene that has not previously been described. The patient was admitted to hospital at the age of 16 days due to salt-losing syndrome with hyperpotassemia. After this episode, he received no treatment for 2 years, when he began to show progressive anorexia, salt avidity, asthenia, cutaneous hyperpigmentation and finally shock, with hypoglycemia, hyponatremia, metabolic acidosis and hyperpotassemia. Puberal development was spontaneous but incomplete. The patient received treatment with testosterone-depot. He reached a definitive testicular volume of 6 ml and pubarche V. His final height is 150 cm (target height 164 cm). Amplification of the DAX-1 gene showed mutation g 2080-2081 insertion in the first position of codon 168, which produces a premature shutdown of protein DAX-1 at position 184.

Identification and characterisation of a novel KCNQ1 mutation in a family with Romano–Ward syndrome

Romano-Ward syndrome (RWS), the autosomal dominant form of the congenital long QT syndrome, is characterised by prolongation of the cardiac repolarisation process associated with ventricular tachyarrhythmias of the torsades de pointes type. Genetic studies have identified mutations in six ion channel genes, KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2 and the accessory protein Ankyrin-B gene, to be responsible for this disorder. Single-strand conformation polymorphism (SSCP) analysis and subsequent DNA sequence analysis have identified a KCNQ1 mutation in a family that were clinically conspicuous due to several syncopes and prolonged QTc intervals in the ECG. The mutant subunit was expressed and functionally characterised in the Xenopus oocyte expression system. A novel heterozygous missense mutation with a C to T transition at the first position of codon 343 (CCA) of the KCNQ1 gene was identified in three concerned family members (QTc intervals: 500, 510 and 530 ms, respectively). As a result, proline 343 localised within the highly conserved transmembrane segment S6 of the KCNQ1 channel is replaced by a serine. Co-expression of mutant (KCNQ1-P343S) and wild-type (KCNQ1) cRNA in Xenopus oocytes produced potassium currents reduced by approximately 92%, while IKs reconstitution experiments with a combination of KCNQ1 mutant, wild-type and KCNE1 subunits yielded currents reduced by approximately 60%. A novel mutation (P343S) identified in the KCNQ1 subunit gene of three members of a RWS family showed a dominant-negative effect on native IKs currents leading to prolongation of the heart repolarisation and possibly increases the risk of malign arrhythmias with sudden cardiac death.

A novel lysozyme mutation Phe57Ile associated with hereditary renal amyloidosis

Variant forms of lysozyme, a ubiquitous bacteriolytic enzyme, are known to lead to hereditary non-neuropathic renal amyloidosis and, so far, three different mutations of the lysozyme gene have been reported. In this study, we report a novel lysozyme variant, Phe57Ile, associated with renal amyloidosis in three patients in one Italian Canadian family. The proband was a 52-year-old woman who developed renal failure at the age of 42 years. Renal biopsy demonstrated replacement of glomeruli by amyloid. Her younger sister and her younger daughter who underwent renal transplantation also had renal amyloidosis. The proband's older daughter and her niece were in good health. To elucidate pathogenesis of this hereditary renal amyloidosis, DNA analyses of the lysozyme gene, including single strand confirmation polymorphism, direct DNA sequence, and restriction fragment length polymorphism analyses, were performed. DNA analyses of the lysozyme gene revealed a T to A transversion at the first position of codon 57 of the lysozyme gene in the proband, her sister, and her affected and unaffected daughters, indicating a replacement of Phe by Ile at residue 57. In addition, DNA sequencing demonstrated a C to A transversion at the second position of codon 70, denoting a replacement of Thr by Asn at residue 70, in the proband's sister and her niece. Thus, the proband's sister is compound heterozygous for the Phe57Ile and Thr70Asn alleles. Distinctive clinical features in patients of this family are nephropathy due to renal amyloidosis. Our results indicate that the novel lysozyme variant Phe57Ile is associated with renal amyloidosis in this family. From our results, a clear relation between the Thr70Asn polymorphism and renal amyloidosis could not be demonstrated.

Biochemical Characterization of a Neuroserpin Variant Associated with Hereditary Dementia

Neuroserpin isolated from inclusion bodies in the brain of a patient with a neurodegenerative disease was characterized biochemically. The protein consisted of residues 20 to 410 of the neuroserpin precursor deduced from its cDNA sequence indicating the entire molecule was deposited. A minor amount started with residue 19 of the precursor, and the carboxyl terminus was heterogeneous ending at residues 405, 407, 409, and 410. Arg was present at position 52. No normal Ser52 was found indicating that only mutant neuroserpin was present in the inclusion bodies. The three potential Asn glycosylation sites all contained carbohydrate. DNA sequence analysis of exons 2 to 9 of the neuroserpin gene in the proband showed the published normal neuroserpin sequence except for the presence of both adenine and cytosine at the first position of codon 52, that indicates heterozygosity for both the normal Ser(AGT) and variant Arg(CGT) at this position in the expressed protein. Restriction fragment length polymorphism analysis of a polymerase chain reaction product from exon 2 revealed the propositus and his affected sibling both were heterozygous for the mutation whereas 100 unaffected controls were negative. Chemical characterization of the variant neuroserpin will significantly enhance the understanding of this protein in both normal physiology and neurodegenerative diseases.

Facilitated diffusion of fructose via the phosphoenolpyruvate/glucose phosphotransferase system of Escherichia coli.

From mutants of Escherichia coli unable to utilize fructose via the phosphoenolpyruvate/glycose phosphotransferase system (PTS), further mutants were selected that grow on fructose as the sole carbon source, albeit with relatively low affinity for that hexose (K(m) for growth approximately 8 mM but with V(max) for generation time approximately 1 h 10 min); the fructose thus taken into the cells is phosphorylated to fructose 6-phosphate by ATP and a cytosolic fructo(manno)kinase (Mak). The gene effecting the translocation of fructose was identified by Hfr-mediated conjugations and by phage-mediated transduction as specifying an isoform of the membrane-spanning enzyme II(Glc) of the PTS, which we designate ptsG-F. Exconjugants that had acquired ptsG(+) from Hfr strains used for mapping (designated ptsG-I) grew very poorly on fructose (V(max) approximately 7 h 20 min), even though they were rich in Mak activity. A mutant of E. coli also rich in Mak but unable to grow on glucose by virtue of transposon-mediated inactivations both of ptsG and of the genes specifying enzyme II(Man) (manXYZ) was restored to growth on glucose by plasmids containing either ptsG-F or ptsG-I, but only the former restored growth on fructose. Sequence analysis showed that the difference between these two forms of ptsG, which was reflected also by differences in the rates at which they translocated mannose and glucose analogs such as methyl alpha-glucoside and 2-deoxyglucose, resided in a substitution of G in ptsG-I by T in ptsG-F in the first position of codon 12, with consequent replacement of valine by phenylalanine in the deduced amino acid sequence.

The Mutation Associated with Hb Peterborough [β111(G13)Val→Phe] Originated from Southern Italy

Hb Peterborough [β111(G13)Val→Phe], an unstable hemoglobin variant with low oxygen affinity, was first described in two patients of Italian origin. This paper reports the first observation of this variant in Campania, Southern Italy, in two unrelated patients suffering from mild anemia. The variant was separated from Hb A by cation exchange chromatography on a high performance liquid chromatographic system with an automated procedure that might be useful for diagnostic purposes. The amino acid replacement, Val→Phe at β111, was assessed by tandem electrospray mass spectrometry analysis, and the corresponding DNA mutation was established as G→T at the first position of codon 111 (GTC→JTC) by polymerase chain reaction amplification techniques. A family study showed that the two original carriers of Hb Peterborough were members of the same family as the proband examined in this study. This finding, and the presence of a second unrelated family carrying Hb Peterborough in Campania, strongly suggests that the DNA mutation associated with this variant originated in Southern Italy.

Characterization and locus assignment of two α-globin variants present in the maltese population: Hb St. Luke's [α95(G2)Pro→Arg] and Hb Setif [α94(G1)Asp→Tyr

Two types of α-globin variants were found in 0.2% of a large number of newborn from Malta. The two hemoglobins were identified from tryptic maps on a Vydac C18 column and by α-globin gene sequencing as Hb St. Luke's (isoelectric point = 7.18 ± 0.017) and Hb Setif (isoelectric point = 7.26 ± 0.010). Hb St. Luke's [α95(G2)Pr→Arg] was found to result from a C→G mutation at the second position of codon 95 on an cd-globin gene, and Hb Setif [α94(Gl) Asp→Tyr] resulted from a G→T mutation at the first position of codon 94 on an α2-globin gene. Quantification of Hb St. Luke's (11.1 ± 1.12%) and Hb Setif (14.7 ± 2.22%) in peripheral blood hemolysates indicated that, in the absence of either an α- or a β-thalassemia allele, the protein products of the α1- and α2-gIobin genes were nearly equal in quantity.

Styrene Oxide Adducts in an Oligodeoxynucleotide Containing the Human N-ras Codon 12: Minor Groove Structures of the R(12,1)- and S(12,1)-α-(N2-Guanyl) Stereoisomers Determined by 1H Nuclear Magnetic Resonance

The R- and S-alpha-(N2-guanyl)styrene oxide (SO) adducts at X5 in d(G1G2C3A4X5G6T7G8G9T10G11).d(C12A13C14C15A16C17C18T19G20C21C22 ), encompassing codon 12 of the human N-ras protooncogene (underlined), were examined using 1H NMR spectroscopy. These were the R(12,1) and S(12,1) adducts, indicating the location of the R or S adduct at the first position of codon 12. These differed from the R- and S(12, 2)-alpha-SO adducts [Zegar, I. S., Setayesh, F. R., DeCorte, B. L., Harris, C. M., Harris, T. M., and Stone, M. P. (1996) Biochemistry 35, 4334-4348] in that the base pair 5' to the lesion was changed from G.C to A.T, while the base pair 3' to the lesion was changed from T.A to G.C. Comparison of the R- and S(12,1) adducts with the R- and S(12,2) adducts allowed the effects of flanking bases on the conformations of the alpha-SO adducts to be examined. This change in flanking base affected the R-SO lesion. The R(12,1) adduct structure was disordered at the adduct site, and a refined structure could not be obtained. NOE and chemical shift data suggested that the styrenyl moiety was oriented in the minor groove and in the 3'-direction from the site of adduction. In contrast, this change in flanking base did not affect the S-SO lesion. The S(12,1) adduct yielded a refined structure, with the styrenyl moiety edgewise in the minor groove and oriented in the 5'-direction relative to the site of adduction. A total of 232 interproton distances, including 13 styrene-DNA distances, were obtained. A total of 12 NOE-restrained molecular dynamics calculations converged with pairwise root-mean-square deviation of 1.10 A. The sixth-root residual index between calculated and experimental NOE intensities was 8.0 x 10(-)2 A. The styrene aromatic protons appeared as three resonances, suggesting rapid rotation. The possibility of a hydrogen bond between the styrene hydroxyl and C18 O2 in the S(12,1) adduct could not be confirmed. This work illustrates the dual roles of stereochemistry and sequence in modulating the properties of guanine N2 alpha-SO adducts.

Hb Köln [&lt;FONT FACE=Symbol&gt;a&lt;/font&gt;2&lt;FONT FACE=Symbol&gt;b&lt;/font&gt;298(FG5) val-met] identified by DNA analysis in a Brazilian family

Hb Köln was identified by DNA analysis in a Brazilian patient. A four-year old Brazilian female, with jaundice since birth, presented an abnormal band, between A2 and S, in hemoglobin electrophoresis on a cellulose acetate membrane, and a band with electrophoretic migration similar to Hb C on agar gel. Thermic instability and isopropanol precipitation tests were positive. Heinz bodies were observed in the patient’s peripheral blood. Sequencing of the three exons of the &lt;FONT FACE="Symbol"&gt;b&lt;/font&gt; globin gene detected a transition from G to A in the first position of codon 98. This alteration does not create or abolish any known restriction site. In this case, confirmation of the mutation was accomplished by allele-specific oligonucleotide hybridization, which is a simple and fast identification method when the clinical data and hematological and electrophoretic patterns are suggestive of Hb Köln.

From essential to beneficial: glycoprotein D loses importance for replication of bovine herpesvirus 1 in cell culture.

Glycoprotein D (gD) of bovine herpesvirus 1 (BHV-1) has been shown to be an essential component of virions involved in virus entry. gD expression in infected cells is also required for direct cell-to-cell spread. Therefore, BHV-1 gD functions are identical in these aspects to those of herpes simplex virus 1 (HSV-1) gD. In contrast, the gD homolog of pseudorabies virus (PrV), although essential for penetration, is not necessary for direct cell-to-cell spread. Cocultivation of cells infected with phenotypically gD-complemented gD- mutant BHV-1/80-221 with noncomplementing cells resulted in the isolation of the cell-to-cell-spreading gD-negative mutant ctcs+BHV-1/80-221, which was present in the gD-null BIV-1 stocks. ctcs+BHV-1/80-221 could be propagated only by mixing infected with uninfected cells, and virions released into the culture medium were noninfectious. Marker rescue experiments revealed that a single point mutation in the first position of codon 450 of the glycoprotein H open reading frame, resulting in a glycine-to-tryptophan exchange, enabled complementation of the gD function for cell-to-cell spread. After about 40 continuous passages of ctcs+BHV-1/80-221-infected cells with noninfected cells, the plaque morphology in the cultures started to change from roundish to comet shaped. Cells from such plaques produced infectious gD- virus, named gD-infBHV-1, which entered cells much more slowly than wild-type BHV-1. In contrast, integration of the gD gene into the genomes of gD-infBHV-1 and ctcs+BHV-1/80-221 resulted in recombinants with accelerated penetration in comparison to wild-type virions. In summary, our results demonstrate that under selective conditions, the function of BHV-1 gD for direct cell-to-cell spread and entry into cells can be compensated for by mutations in other viral (glyco)proteins, leading to the hypothesis that gD is involved in formation of penetration-mediating complexes in the viral envelope of which gH is a component. Together with results for PrV, varicella-zoster virus, which lacks a gD homolog, and Marek's disease virus, whose gD homolog is not essential for infectivity, our data may open new insights into the evolution of alphaherpesviruses.