Triplet Deletion Research Articles

Pervasive fungicide resistance in Botrytis from strawberry in Norway: Identification of the grey mould pathogen and mutations

AbstractControl of grey mould, caused by Botrytis spp., is a major challenge in open field strawberry production. Botrytis was isolated from plant parts collected from 19 perennial strawberry fields with suspected fungicide resistance in the Agder region of Norway in 2016. Resistance to boscalid, pyraclostrobin and fenhexamid was high and found in 89.1%, 86.0% and 65.4% of conidia samples, respectively. Multiple fungicide resistance was common; 69.6% of conidia samples exhibited resistance to three or more fungicides. Botrytis group S and B. cinerea sensu stricto isolates were obtained from 19 and 16 fields, respectively. The sdhB, cytb, erg27 and mrr1 genes of a selection of isolates were examined for the presence of mutations known to confer fungicide resistance to boscalid, pyraclostrobin, fenhexamid and pyrimethanil plus fludioxonil, respectively. Allele‐specific PCR assays were developed for efficient detection of resistance‐conferring mutations in cytb. Among B. cinerea isolates, 84.7%, 86.3% and 61.3% had resistance‐conferring mutations in sdhB, cytb and erg27, respectively. A triplet deletion in mrr1, resulting in ΔL497, commonly associated with the multidrug resistance phenotype MDR1h, was detected in 29.2% of Botrytis group S isolates. High frequencies of resistance to several fungicides were also detected in Botrytis from both imported and domestically produced strawberry transplants. Fungicide resistance frequencies were not different among fields grouped by level of grey mould problem assessed by growers, indicating factors other than fungicide resistance contributed to control failure, a fact that has important implications for future management of grey mould.

Incidental diagnosis of leukocyte adhesion deficiency type II following ABO typing

Individuals with the Bombay phenotype (Oh) in the ABO blood group system do not express the H, A, and B antigens but have no clinical symptoms. Bombay phenotype with clinical symptoms has been described in leukocyte adhesion deficiency type II (LAD II), a fucosylation disorder caused by mutations in SLC35C1. Only few LAD II patients have been described so far. Here we describe an additional patient, a 22-year old male, born to unrelated parents, presenting with inflammatory skin disease, periodontitis, growth, and mental retardation, admitted to the department of dentistry for treatment under general anesthesia. Pre-operative routine investigations revealed the presence of the Bombay phenotype (Oh). Genomic sequencing identified two novel triplet deletions of the SLC35C1 gene. Functional investigations confirmed the diagnosis of LAD II. Therapy with oral fucose led to the disappearance of the chronic skin infections and improvements in behavior and attention span.

Genome-Wide DNA Alterations in X-Irradiated Human Gingiva Fibroblasts.

While ionizing radiation (IR) is a powerful tool in medical diagnostics, nuclear medicine, and radiology, it also is a serious threat to the integrity of genetic material. Mutagenic effects of IR to the human genome have long been the subject of research, yet still comparatively little is known about the genome-wide effects of IR exposure on the DNA-sequence level. In this study, we employed high throughput sequencing technologies to investigate IR-induced DNA alterations in human gingiva fibroblasts (HGF) that were acutely exposed to 0.5, 2, and 10 Gy of 240 kV X-radiation followed by repair times of 16 h or 7 days before whole-genome sequencing (WGS). Our analysis of the obtained WGS datasets revealed patterns of IR-induced variant (SNV and InDel) accumulation across the genome, within chromosomes as well as around the borders of topologically associating domains (TADs). Chromosome 19 consistently accumulated the highest SNVs and InDels events. Translocations showed variable patterns but with recurrent chromosomes of origin (e.g., Chr7 and Chr16). IR-induced InDels showed a relative increase in number relative to SNVs and a characteristic signature with respect to the frequency of triplet deletions in areas without repetitive or microhomology features. Overall experimental conditions and datasets the majority of SNVs per genome had no or little predicted functional impact with a maximum of 62, showing damaging potential. A dose-dependent effect of IR was surprisingly not apparent. We also observed a significant reduction in transition/transversion (Ti/Tv) ratios for IR-dependent SNVs, which could point to a contribution of the mismatch repair (MMR) system that strongly favors the repair of transitions over transversions, to the IR-induced DNA-damage response in human cells. Taken together, our results show the presence of distinguishable characteristic patterns of IR-induced DNA-alterations on a genome-wide level and implicate DNA-repair mechanisms in the formation of these signatures.

The evolution of multiple-insecticide resistance in UK populations of tomato leafminer, Tuta absoluta.

The tomato leafminer, Tuta absoluta, is an economically important pest of tomatoes in Europe, Africa, Asia and South America. In the UK this species is controlled using an integrated pest management (IPM) programme which incorporates the insecticides spinosad and chlorantraniliprole. In response to UK grower concerns of loss of efficacy of these compounds at certain sites, insecticide bioassays were performed on five populations collected from four commercial glasshouses and potential mechanisms of resistance investigated. We observed high levels of resistance to spinosad in four of the strains, and in two of these tolerance to chlorantraniliprole. Selection of one of these strains with chlorantraniliprole rapidly resulted in a line exhibiting potent resistance to this compound. Sequencing of messenger RNA encoding the nicotinic acetylcholine receptor (nAChR) α6 subunit, target of spinosad, revealed Taα6 transcripts in the spinosad-resistant strains that lack exon 4 and encode a highly truncated protein, or contain a triplet deletion in the predicted first transmembrane domain resulting in the loss of a highly conserved amino acid. Sequencing of the ryanodine receptor gene, encoding the target of diamide insecticides, of the chlorantraniliprole-selected line revealed an amino acid substitution (G4903V) that has been previously linked to diamide resistance in populations of T. absoluta in the Mediterranean and South America. Taken together our results reveal emerging resistance in UK populations of T. absoluta to two of the most important insecticides used as part of IPM, with significant implications for the control of this species in the UK. © 2019 Society of Chemical Industry.

Functional analysis of a triplet deletion in the gene encoding the sodium glucose transporter 3, a potential risk factor for ADHD.

Sodium-glucose transporters (SGLT) belong to the solute carrier 5 family, which is characterized by sodium dependent transport of sugars and other solutes. In contrast, the human SGLT3 (hSGLT3) isoform, encoded by SLC5A4, acts as a glucose sensor that does not transport sugar but induces membrane depolarization by Na+ currents upon ligand binding. Whole-exome sequencing (WES) of several extended pedigrees with high density of attention-deficit/hyperactivity disorder (ADHD) identified a triplet ATG deletion in SLC5A4 leading to a single amino acid loss (ΔM500) in the hSGLT3 protein imperfectly co-segregating with the clinical phenotype of ADHD. Since mutations in homologous domains of hSGLT1 and hSGLT2 were found to affect intestinal and renal function, respectively, we analyzed the functional properties of hSGLT3[wt] and [ΔM500] by voltage clamp and current clamp recordings from cRNA-injected Xenopus laevis oocytes.The cation conductance of hSGLT3[wt] was activated by application of glucose or the specific agonist 1-desoxynojirimycin (DNJ) as revealed by inward currents in the voltage clamp configuration and cell depolarization in the current clamp mode. Almost no currents and changes in membrane potential were observed when glucose or DNJ were applied to hSGLT3[ΔM500]-injected oocytes, demonstrating a loss of function by this amino acid deletion in hSGLT3. To monitor membrane targeting of wt and mutant hSGLT3, fusion constructs with YFP were generated, heterologously expressed in Xenopus laevis oocytes and analyzed for membrane fluorescence by confocal microscopy. In comparison to hSGLT3[wt] the fluorescent signal of mutant [ΔM500] was reduced by 43% indicating that the mutant phenotype might mainly result from inaccurate membrane targeting. As revealed by homology modeling, residue M500 is located in TM11 suggesting that in addition to the core structure (TM1-TM10) of the transporter, the surrounding TMs are equally crucial for transport/sensor function.In conclusion, our findings indicate that the deletion [ΔM500] in hSGLT3 inhibits membrane targeting and thus largely disrupts glucose-induced sodium conductance, which may, in interaction with other ADHD risk-related gene variants, influence the risk for ADHD in deletion carriers.

N-terminal truncation contributed to increasing thermal stability of mannanase Man1312 without activity loss.

The disordered residues on distal loops affect the molecular structural stability and on some occasions have regulatory roles in catalytic reaction. To increase understanding of the influence of distal residue mutation, this study explored the thermostability and enzymatic activity of mannanase Man1312 deletion mutants. The focus was on residues located on the N-terminal region because they are more disordered and changeable. The effects of N-terminal truncation on enzymatic activity and thermal dynamics were investigated by spectrophotometry, circular dichroism and differential scanning calorimetry assays. The deletion mutants on V3, N7 and Q11 showed a marked increase in stability, while the enzymatic activity was significantly improved when triplet deletion was carried out. Triplet deletion MandVNQ showed around double the stability of its corresponding single-site and double-site deletion mutants. The Tm value of MandVNP was about 8 °C higher than that of Man1312. MandVNP had improved characteristics of Topt by 10 °C, t1/2 by 10 min and catalytic activity by 11% in comparison with Man1312. Analysis of spectra and modeling showed that MandVNQ had increased helix and strand contents. N-terminal truncation had positive effects on the thermostability and activity of mannanase.

A Label-Free, Electrochemical SERS-Based Assay for Detection of DNA Hybridization and Discrimination of Mutations

A label-free, surface-enhanced Raman spectroscopy-based assay for detecting DNA hybridization at an electrode surface and for distinguishing between mutations in DNA is demonstrated. Surface-immobilized DNA is exposed to a binding agent that is selective for dsDNA and acts as a reporter molecule. Upon application of a negative potential, the dsDNA denatures into its constituent strands, and the changes in the spectra of the reporter molecule are monitored. This method has been used to distinguish between a wild-type, 1653C/T single-point mutation and ΔF508 triplet deletion in the CFTR gene. The use of dsDNA-selective binding agents as reporter molecules in a discrimination assay removes the burden of synthetically modifying the target to be detected, while retaining flexibility in the choice of the reporter molecule.

Local sequence determinants of two in-frame triplet deletion/duplication hotspots in the RHD/RHCEgenes

Different types of human gene mutation can vary in size quite dramatically (e.g., single nucleotide substitutions vs. copy number variations), but what they all have in common is that their occurrence is often closely bound up with specific characteristics of the local DNA sequence environment [1]. Here, we highlight the importance of local sequence features that underlie the two in-frame triplet deletion/duplication hotspots in the cis-linked, highly homologous RHD and RHCE paralogs. The first hotspot refers to an 8-bp sequence tract in exon 1 of the RHD and RHCE genes, in which three different variants were reported (Figure ​(Figure1a)1a) [2-4]. The first variant is a deletion of one of two juxtaposed CTC triplets in the RHD gene, which gives rise to an in-frame deletion of a single amino acid, Leu27 [2]. The second variant is identical to the first but occurred at the analogous location in the RHCE gene [3]. Henceforth, we shall employ the term ‘deduplication’ [5], which emphasizes the identity of the deleted sequence and the sequence immediately abutting the site of the deletion, to describe this particular type of microdeletion (<21 bp in length in accordance with Ball et al. [6]). Deduplication accounts for a significant proportion of disease-causing microdeletions; indeed, for microdeletion events of 2–5 bp, 38 % were found to be deduplications [6]. Replication slippage is currently regarded as the major mechanism underlying the generation of deduplications: the primer strand containing the newly synthesized first direct repeat dissociates from the template strand and then misaligns (slipping forward) at the second direct repeat; continued DNA synthesis then leads to the deletion of one of the two direct repeats. It should be pointed out that while direct repeats are a prerequisite for replication slippage, they are certainly not the sole determinant of this mutational mechanism. In this regard, we noted that the two CTC triplets together constitute a DNA polymerase arrest site (consensus sequence WGGAG, where W = A or T [7]) (Figure ​(Figure1a).1a). It is therefore tempting to speculate that a combination of these two sequence features could have served to make this short region particularly prone to replication slippage. Figure 1 The three currently known in-frame triplet deletion/duplication variants in exon 1 of the RHD/RHCE genes. Their underlying mutational mechanisms are also shown. (a) Illustration of the identical deletion of one of two neighboring CTC repeats (in bold ... Recently, Pereira and colleagues reported the first in-frame triplet duplication in the RHD gene; this duplication affected the same short region as the aforementioned two deduplications in exon 1 (Figure ​(Figure1a)1a) [4]. As pointed out by the original authors, this duplication could have resulted from either a duplication of c.74-76TTC or c.75_77TCT. These authors emphasized the importance of a DNA motif (i.e., TTCTC that was identified by analogy to previously reported deletion-predisposing DNA motifs in the RHD gene [9]) in generating this duplication but did not provide a model to explain how this duplication could have been generated. Given that the sequence tract in question is prone to replication slippage, we surmised that this duplication might also be explicable in terms of such a mechanism. Indeed, as illustrated in Figure ​Figure1b,1b, it can be readily explained by the model of serial replication slippage [8], invoking one step of forward slippage and one step of backward slippage. The second hotspot refers to a 63-bp region of exon 5 in the RHD and RHCE genes, in which four in-frame triplet deletions (c.644_646delTCT [3], c.684_686delGAG, and c.705_707delAGA [9] in RHD; c.685_687delAGA [10] in RHCE) were reported (Figure ​(Figure2).2). Several distinct DNA repeats or motifs (e.g., GAGAA and GAAGA) have previously been implicated in the generation of three of these four variants [9]. A comparative evaluation of the four variants led us to propose a consensus motif RAGAA (R = A or G) (Figure ​(Figure2).2). Since only the c.644_646delTCT variant can be explained in terms of replication slippage, it may be that RAGAA is associated with a recombination-predisposing activity that is distinct from the DNA polymerase arrest site WGGAG. In other words, the different local sequence contexts in exons 1 and 5 of RHD and RHCE could predispose to subtly different mutational processes. Figure 2 The three 3-bp deletion variants in exon 5 of RHD. The deletions are highlighted in bold. The underlined sequences refer to the consensus motif RAGAA or its complement. The c.685_687AGA deletion in RHCE [10] is not shown.

A novel ABO allele caused by an in‐frame triplet deletion in ABO gene

Vox SanguinisVolume 103, Issue 4 p. 362-363 LETTER A novel ABO allele caused by an in-frame triplet deletion in ABO gene L. Tian, L. Tian Department of Blood Immunology, Institute of Blood Transfusion, Chinese, Academy of Medical Sciences, Chengdu, Sichuan, ChinaSearch for more papers by this authorZ.-Q. Yao, Z.-Q. Yao Department of Blood Immunology, Institute of Blood Transfusion, Chinese, Academy of Medical Sciences, Chengdu, Sichuan, ChinaSearch for more papers by this authorW.-C. Fan, W.-C. Fan Mianyang Red Cross Blood Center, Mianyang, Sichuan, ChinaSearch for more papers by this authorH. Li, H. Li Department of Blood Immunology, Institute of Blood Transfusion, Chinese, Academy of Medical Sciences, Chengdu, Sichuan, ChinaSearch for more papers by this authorN. Song, N. Song Department of Blood Immunology, Institute of Blood Transfusion, Chinese, Academy of Medical Sciences, Chengdu, Sichuan, ChinaSearch for more papers by this author L. Tian, L. Tian Department of Blood Immunology, Institute of Blood Transfusion, Chinese, Academy of Medical Sciences, Chengdu, Sichuan, ChinaSearch for more papers by this authorZ.-Q. Yao, Z.-Q. Yao Department of Blood Immunology, Institute of Blood Transfusion, Chinese, Academy of Medical Sciences, Chengdu, Sichuan, ChinaSearch for more papers by this authorW.-C. Fan, W.-C. Fan Mianyang Red Cross Blood Center, Mianyang, Sichuan, ChinaSearch for more papers by this authorH. Li, H. Li Department of Blood Immunology, Institute of Blood Transfusion, Chinese, Academy of Medical Sciences, Chengdu, Sichuan, ChinaSearch for more papers by this authorN. Song, N. Song Department of Blood Immunology, Institute of Blood Transfusion, Chinese, Academy of Medical Sciences, Chengdu, Sichuan, ChinaSearch for more papers by this author First published: 27 March 2012 https://doi.org/10.1111/j.1423-0410.2012.01607.xCitations: 4 Ning Song Department of Blood Immunology Institute of Blood Transfusion Chinese Academy of Medical Sciences Chengdu Sichuan 610052 China E-mail: watercrowd@163.com Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume103, Issue4November 2012Pages 362-363 RelatedInformation

Multilocus Sequence Typing Reveals Relevant Genetic Variation and Different Evolutionary Dynamics among Strains of Xanthomonas arboricola pv. juglandis

Forty-five Xanthomonas arboricola pv. juglandis (Xaj) strains originating from Juglans regia cultivation in different countries were molecularly typed by means of MultiLocus Sequence Typing (MLST), using acnB, gapA, gyrB and rpoD gene fragments. A total of 2.5 kilobases was used to infer the phylogenetic relationship among the strains and possible recombination events. Haplotype diversity, linkage disequilibrium analysis, selection tests, gene flow estimates and codon adaptation index were also assessed. The dendrograms built by maximum likelihood with concatenated nucleotide and amino acid sequences revealed two major and two minor phylotypes. The same haplotype was found in strains originating from different continents, and different haplotypes were found in strains isolated in the same year from the same location. A recombination breakpoint was detected within the rpoD gene fragment. At the pathovar level, the Xaj populations studied here are clonal and under neutral selection. However, four Xaj strains isolated from walnut fruits with apical necrosis are under diversifying selection, suggesting a possible new adaptation. Gene flow estimates do not support the hypothesis of geographic isolation of the strains, even though the genetic diversity between the strains increases as the geographic distance between them increases. A triplet deletion, causing the absence of valine, was found in the rpoD fragment of all 45 Xaj strains when compared with X. axonopodis pv. citri strain 306. The codon adaptation index was high in all four genes studied, indicating a relevant metabolic activity.

Association betweenFOXP2gene and speech sound disorder in Chinese population

FOXP2 was described as the first gene relevant to human speech and language disorders. The main objective of this study was to compare the distribution of FOXP2 gene polymorphisms between patients with speech sound disorder and healthy controls. Five FOXP2 polymorphisms, rs923875, rs2396722, rs1852469, rs17137124 and rs1456031, were analyzed in 150 patients with speech sound disorder according to DSM-IV, as well as in 140 healthy controls. Coding exons for key domains of FOXP2 were also sequenced in all the patients. Significant differences in the genotype (P = 0.001) and allele (P = 0.0025) frequencies of rs1852469 (located 5' upstream of the ATG initiator codon) were found between patients and controls. The excess of the T allele in the patients group remained significant after Bonferroni correction (P = 0.0126). Further investigations revealed a risk haplotype: rs2396722T/+rs1852469T. Our screening of key domains did not detect any point mutations in this sample. But we detected heterozygous triplet deletion of the glutamine-encoding region of exon 5 that alter FOXP2 protein sequence in five probands. These changes are predicted to yield a polyglutamine tract reduction from 40 to 39 consecutive glutamines. Our data support a possible role of FOXP2 in the vulnerability to speech sound disorder, which adds further evidence to implicate this gene in speech and language functions.

Novel UBE2B-associated polymorphisms in an azoospermic/oligozoospermic population

To assess whether abnormalities exist in the UBE2B gene in a population of infertile human males, and to establish biologic plausibility of any discovered mutations. We carried out polymerase chain reaction (PCR) amplification and sequence analysis of the 5'-untranslated region and six exons of the UBE2B gene, including flanking intronic regions, in a group of fertile and infertile men. Following the identification of a putative promoter region that contained single or dual triplet deletions within a 10-CGG repeat island, we evaluated the binding affinity of these identified polymorphisms as compared to the wild-type sequence to transcription factor SP1 using a DNA-protein gel shift assay. There was a novel exonic single nucleotide polymorphism (SNP) noted in exon 4 in 5% of infertile men. In silico 3D modeling of the altered protein showed an innocuous isoleucine for valine substitution. There were no mutations noted within any of the other exons. Three novel intronic SNPs were identified within the fertile group, and seven novel intronic SNPs identified in the infertile group. The DNA-protein gel shift assay noted that both single CGG deletion and double CGG deletion bands had approximately twice the binding affinity compared to the wild-type for SP1. The negative control confirmed no non-specific protein binding. By themselves, a single or double CGG deletion is unlikely to pose biologic significance. However, such deletions in this suspected promoter region are associated with increased binding affinity for SP1, and might represent one of several factors required for alteration of UBE2B gene expression.

Single and dual CGG deletions in the 5' UTR of the UBE2B gene increases binding to transcription factor SP1, and may be a cause of human male infertility

The UBE2B gene is one of many genes involved in human spermatogenesis, the disruption of which has not previously been described as a factor in human male infertility. In the male murine model however, the UBE2B mouse knockout is infertile due to early spermatogenic arrest. Previously, we have demonstrated that while Exons 1–6 of the UBE2B gene are highly conserved in a group of 96 men with severe oligospermia or non-obstructive azoospermia, there is a 10 CGG island within a suspected promoter region of the UBE2B gene that is altered in 4% of this same infertile male population, specifically with single or dual CGG triplet deletions.

In‐frame triplet deletions in RHD alter the D antigen phenotype

The deletion of three adjacent nucleotides in an exon may cause the lack of a single amino acid, while the protein sequence remains otherwise unchanged. Only one such in-frame deletion is known in the two RH genes, represented by the RHCE allele ceBP expressing a "very weak e antigen." Blood donor samples were recognized because of discrepant results of D phenotyping. Six samples came from Switzerland and one from Northern Germany. The molecular structures were determined by genomic DNA nucleotide sequencing of RHD. Two different variant D antigens were explained by RHD alleles harboring one in-frame triplet deletion each. Both single-amino-acid deletions led to partial D phenotypes with weak D antigen expression. Because of their D category V-like phenotypes, the RHD(Arg229del) allele was dubbed DVL-1 and the RHD(Lys235del) allele DVL-2. These in-frame triplet deletions are located in GAGAA or GAAGA repeats of the RHD exon 5. Partial D may be caused by a single-amino-acid deletion in RhD. The altered RhD protein segments in DVL types are adjacent to the extracellular loop 4, which constitutes one of the most immunogenic parts of the D antigen. These RhD protein segments are also altered in all DV, which may explain the similarity in phenotype. At the nucleotide level, the triplet deletions may have resulted from replication slippage. A total of nine amino acid positions in an Rhesus protein may be affected by this mechanism.

Contingency Locus in ctsR of Listeria monocytogenes Scott A: a Strategy for Occurrence of Abundant Piezotolerant Isolates within Clonal Populations

In a recent study we demonstrated that a high-hydrostatic-pressure-tolerant isolate of Listeria monocytogenes lacks a codon in the class 3 heat shock regulator gene ctsR. This mutation in the region that encodes four consecutive glycines was directly responsible for the observed piezotolerance, increased stress resistance, and reduced virulence. The aim of the present study was to determine whether mutations in ctsR are frequently associated with piezotolerance in L. monocytogenes. Wild-type cultures of L. monocytogenes were therefore exposed to 350 MPa for 20 min, and the piezotolerance of individual surviving isolates was assessed. This rendered 33 isolates with a stable piezotolerant phenotype from a total of 84 survivors. Stable piezotolerant mutants were estimated to be present in the initial wild-type population at frequencies of >10(-5). Subsequent sequencing of the ctsR gene of all stable piezotolerant isolates revealed that two-thirds of the strains (i.e., n = 21) had mutations in this gene. The majority of the mutations (16 of 21 strains) consisted of a triplet deletion in the glycine-encoding region of ctsR, identical to what was found in our previous study. Interestingly, 2 of 21 mutants contained a codon insertion in this repeat region. The remaining three stable piezotolerant strains showed a 19-bp insertion in the glycine repeat region, a 16-bp insertion downstream of the glycine repeat area (both leading to frameshifts and a truncated ctsR), and an in-frame 114-bp deletion encoding a drastically shortened carboxy terminus of CtsR. In four instances it was not possible to generate a PCR product. A piezotolerant phenotype could not be linked to mutations in ctsR in 8 of 33 isolates, indicating that other thus-far-unknown mechanisms also lead to stable piezotolerance. The present study highlights the importance of ctsR in piezotolerance and stress tolerance of L. monocytogenes, and it demonstrates that short-sequence repeat regions contribute significantly to the occurrence of a piezotolerant and stress-tolerant subpopulation within L. monocytogenes cultures, thus playing an important role in survival.

Characterization of the triplet repeats in the central domain of the γ 134·5 protein of herpes simplex virus 1

The gamma134.5 protein of herpes simplex virus 1 (HSV-1) consists of an amino-terminal domain, a central domain with triplet repeats (Ala-Thr-Pro) and a carboxyl-terminal domain. The triplet repeats are a unique feature of the gamma134.5 protein encoded by HSV-1, but the number of repeats varies among different strains. Notably, the central domain containing the triplet repeats is implicated in neuroinvasion. In this report, it has been shown that partial or full deletion of triplet repeats, i.e. from ten to either three or zero, in the gamma134.5 protein has no effect on the virus response to interferon. The triplet deletion mutants replicate efficiently in CV-1 and mouse 10T1/2 cells. However, in mouse 3T6 cells, these mutants grow with delayed growth kinetics. This decrease in growth, compared with wild-type HSV-1(F), does not result from failure of the virus to suppress the RNA-dependent protein kinase response, but rather from a delay in virus release or egress. Accordingly, these mutant viruses are predominantly present within infected cells. These results indicate that deletions in the central domain of the gamma134.5 protein impair virus egress, but not virus response to interferon.

Rapid and sensitive detection of internal tandem duplication and activating loop mutations of FLT3

Mutations of the FLT3 gene, a receptor tyrosine kinase, are the most frequent genetic alteration reported in acute myeloid leukaemia, with internal tandem duplications (ITD) or mutations within the activating loop (AL) reported at a frequency of around 24% and 6%, respectively. ITD mutations have associated with a poor prognosis. In this study we have used polymerase chain reaction (PCR), combined with restriction enzyme digestion for the detection of AL mutations, with the DNA products separated on the Agilent 2100 Bioanalyser using a DNA-500 kit. This analysis enabled the rapid identification of mutations in FLT3, approximate sizing of the ITD, an estimate of the proportion of mutant RNA and in some cases, specific heteroduplex patterns associated with triplet deletions. Our data shows that approximately 16% of the patients examined had an ITD mutation and over 13% had a mutation in the AL including triplet deletions involving codons 835/836 and point mutations in codon D839. Based on the sensitivity and speed of the bioanalyser, we suggest that this method is invaluable and provides an improvement to the current use of agarose gels for the analysis of FLT3 PCR products.

A new BRAF gene mutation detected in a case of a solid variant of papillary thyroid carcinoma

BRAF gene mutations have been frequently detected in papillary thyroid carcinoma (PTC). Moreover, there is a close association between the type of mutation and the PTC histotype: BRAF(V600E) is associated with conventional PTC and with histological variants of PTC displaying a prominent papillary growth pattern, whereas BRAF(K601E) is associated with the follicular variant of PTC. We report the detection of a novel BRAF triplet deletion in a case of PTC displaying a predominantly solid growth pattern. The deletion leads to the replacement of a valine and a lysine by a glutamate in the BRAF activation segment (BRAF(VK600-1E)), thus mimicking partially the 2 BRAF mutations previously described. Our study reinforces the existence of a close relationship between the occurrence of some types of BRAF mutation and some PTC histotypes. The genetic study of more cases of the solid variant of PTC is necessary to find whether there exists a significant association between the occurrence of BRAF(VK600-1E) and such PTC histotype.

FROM PROTEIN SYNTHESIS TO GENETIC INSERTION

In 1946, (14)C-cyanide made its appearance as an offshoot of the Atomic Energy Program. Our colleague Robert Loftfield built it into (14)C-alanine by the Strecker synthesis, and a lusty program directed toward uncovering the unknown mechanism of protein synthesis grew out of this beginning. The necessity for an undiscovered series of steps and enzymes was soon evident. A cell free system was developed, and a succession of components necessary for this new pathway tumbled out. ATP dependence, amino acid activation, the ribosome as the site of polypeptide formation, discovery of tRNA as the translation molecule linking the gene and protein sequence, and GTP as the essential energy ingredient in peptide chain extension all appeared from our laboratory within the next decade. A little later the AP(4)N family, whose functions remain imperfectly defined, of intracellular molecules was discovered. Isolation of specific species of RNA became a high priority, and we sequenced a small segment of the 3' end of the Rous sarcoma virus, just inside the poly(A) tail, at the same time the Gilbert group at Harvard was sequencing the 5' end. The sequence identity and polarity of the two ends suggested a circular intermediate in replication and predicted correctly that a synthetic antisense oligonucleotide targeted against this sequence might be a specific inhibitor of replication. More recently, we have evolved a technique that appears to achieve a trinucleotide insertion into tissue culture cells bearing a specific Delta508 mRNA triplet deletion, resulting in phenotypic reversion in the tissue culture.

Deletion of arginine codon 229 in the Rhce gene alters e and f but not c antigen expression.

Rh CcEe antigens occur as ce, Ce, cE, or CE alleles in the RBC membrane. Their epitope structures and the location of their cis interacting products remain to be defined. A rare blood sample from a white male whose parents are first cousins was identified. Hemagglutination was performed using standard methods. RH structure and genotype was assessed by Southern blots. Rh transcripts were obtained by gene-specific RT-PCR and sequenced. The mutation was verified by genomic PCR assays. The donor's RBCs typed D+C-c+E-e-f(Rh6)- with a normal c dose, suggesting the Dc- phenotype. Further tests revealed a weak and qualitatively altered e expression. Southern blots indicated a genotype of Dce/dce without other gross changes. RT-PCR detected a triplet deletion (Delta685AGA687) in the Rhce gene that specifies codon 229 for arginine (Arg229). Sequencing of the region around the mutated exon 5 confirmed the donor to be homozygous for the AGA deletion. Arg229 is invariant on external loop 4 and close to the Ala226Pro change specific for e/E polymorphism. The qualitative and quantitative alteration of e antigen defines Arg229 as a crucial component for e/E epitope presentation. Given a normal dose of c antigen, the disruption of f (Rh6) by Arg229 deletion suggests that external loop 4 is a major structural element contributing to the expression of RHCE cis interacting antigenic products.