POLH Research Articles

A Case of Corneal Dysplasia with Identification of POLH Gene Variants in Xeroderma Pigmentosum

A Case of Corneal Dysplasia with Identification of <i>POLH</i> Gene Variants in Xeroderma Pigmentosum

Morphological, genetic and biological characterisation of a novel alphabaculovirus isolated from Cryptophlebia peltastica (Lepidoptera: Tortricidae)

Cryptophlebia peltastica is an agricultural pest of litchis and macadamias in South Africa with phytosanitary status for certain markets. Current control methods rely on chemical, cultural and classical biological control. However, a microbial control option has not been developed. An Alphabaculovirus from C. peltastica was recovered from a laboratory reared colony and morphologically characterised by transmission electron microscopy (TEM). Analysis of occlusion bodies indicated a single NPV (SNPV) varying in size from 421 to 1263 nm. PCR amplification and sequencing of the polh gene region using universal primers followed by BLAST analysis revealed a 93% similarity to a partial polh gene sequence from Epinotia granitalis NPV. Further genetic characterisation involving single restriction endonuclease (REN) digestion of genomic DNA was carried out to generate profiles for comparison against other baculovirus species and potential new isolates of the same virus. The complete genome of the virus was sequenced, assembled and analysed for a more comprehensive genetic analysis. The genome was 115728 base pairs (bp) in length with a GC content of 37.2%. A total of 126 open reading frames (ORFs) were identified with minimal overlap and no preference in orientation. Bioassays were used to determine the virulence of the NPV against C. peltastica. The NPV was virulent against C. peltastica with an LC50 value of 6.46 × 103 OBs/ml and an LC90 value of 2.46 × 105 OBs/ml, and time mortality ranging between 76.32 h and 93.49 h. This is the first study to describe the isolation and genetic characterisation of a novel SNPV from C. peltastica, which has potential for development into a biopesticide for the control of this pest in South Africa.

Baculovirus Kimura two-parameter species demarcation criterion is confirmed by the distances of 38 core gene nucleotide sequences.

Kimura two-parameter nucleotide distance comparisons based on polyhedrin/granulin (polh/gran), late expression factor 8 (lef-8) and late expression factor 9 (lef-9) are a widely applied method for species demarcation for lepidopteran-specific baculoviruses. Baculoviruses are considered to belong to the same species when a pairwise distance threshold of 0.015 is not exceeded and are considered as possibly belonging to the same species with a distance of up to 0.050. In the present work this method was revised and extended for 172 entirely sequenced lepidopteran, hymenopteran and dipteran baculovirus genomes by applying the nucleotide sequences of all 38 known baculovirus core genes for pairwise distance calculations. On the basis of this large dataset, the previously established standard thresholds for baculovirus species demarcation were adjusted for pairwise nucleotide distances estimated from the alignments of all 38 core genes. With the newly applied thresholds for the 38 core-gene dataset, a more sophisticated Kimura two-parameter method was established, avoiding the possible influence of the chimerical polh gene of the Autographa californica multiple nucleopolyhedrovirus. Based on the new dataset, the present classification of baculovirus species was confirmed. Thereby the Kimura two-parameter method for baculovirus demarcation was extended to include the information from all 38 Baculoviridae core genes, which represent the established standard information for baculovirus phylogeny to date.

Characterisation of three Alphabaculovirus isolates from the gypsy moth, Lymantria dispar dispar (Lepidoptera: Erebidae), in Turkey

ABSTRACTIn this study, Lymantria dispar dispar larvae, collected from three different localities in Turkey, were examined for the presence of inclusion bodies under phase contrast and electron microscopes. Inclusion bodies from infected larvae were subjected to polymerase chain reaction using the conserved primers for polyhedrin (polh), late expression factor 8 (lef-8) and late expression factor 9 (lef-9) genes. Sequence analysis confirmed that larvae collected from the three different localities contained multiple nucleopolyhedrosis viruses (MNPVs). These isolates were designated LdMNPV-T1, LdMNPV-T2 and LdMNPV-T3. Phylogenetic analyses of these isolates were performed using target genes polh, lef-8 and lef-9. Restriction endonuclease analysis of the three geographic isolates with EcoRI and PstI enzymes demonstrated some differences existed among the isolates. According to the EcoRI profile, the mean estimated size for the complete genome of each isolate (LdMNPV-T1, LdMNPV-T2 and LdMNPV-T3) was calculated to be approximately 170, 153 and 170 kb, respectively. Insecticidal activities of each isolate were tested on L. d. dispar larvae using four different viral concentrations between 103 and 106 OBs/ml. Results showed that the mortalities for LdMNPV-T1, -T2 and -T3 ranged between 13–53%, 47–100% and 46–93%, respectively. The LC50 and LC95 values of LdMNPV-T2 were not significantly different from the respective corresponding values of the other two isolates. However, isolate LdMNPV-T2 killed larvae with a LC50 value that was lower than the other two isolates. Our results suggested there are promising LdMNPV isolates in Turkey that can be used for microbial control of L. d. dispar larvae.

Diagnosis of Xeroderma pigmentosum variant in a young patient with two novel mutations in the POLH gene.

We describe the characterization of Xeroderma Pigmentosum variant (XPV) in a young Caucasian patient with phototype I, who exhibited a high sensitivity to sunburn and multiple cutaneous tumors at the age of 15 years. Two novel mutations in the POLH gene, which encodes the translesion DNA polymerase η, with loss of function due to two independent exon skippings, are reported to be associated as a compound heterozygous state in the patient. Western blot analysis performed on proteins from dermal fibroblasts derived from the patient and analysis of the mutation spectrum on immunoglobulin genes produced during the somatic hypermutation process in his memory B cells, show the total absence of translesion polymerase η activity in the patient. The total lack of Polη activity, necessary to bypass in an error-free manner UVR-induced pyrimidine dimers following sun exposure, explains the early unusual clinical appearance of this patient.

The Binding Sites of miR-619-5p in the mRNAs of Human and Orthologous Genes

BackgroundNormally, one miRNA interacts with the mRNA of one gene. However, there are miRNAs that can bind to many mRNAs, and one mRNA can be the target of many miRNAs. This significantly complicates the study of the properties of miRNAs and their diagnostic and medical applications.ResultsThe search of 2,750 human microRNAs (miRNAs) binding sites in 12,175 mRNAs of human genes using the MirTarget program has been completed. For the binding sites of the miR-619-5p the hybridization free energy of the bonds was equal to 100% of the maximum potential free energy. The mRNAs of 201 human genes have complete complementary binding sites of miR-619-5p in the 3’UTR (214 sites), CDS (3 sites), and 5’UTR (4 sites). The mRNAs of CATAD1, ICA1L, GK5, POLH, and PRR11 genes have six miR-619-5p binding sites, and the mRNAs of OPA3 and CYP20A1 genes have eight and ten binding sites, respectively. All of these miR-619-5p binding sites are located in the 3’UTRs. The miR-619-5p binding site in the 5’UTR of mRNA of human USP29 gene is found in the mRNAs of orthologous genes of primates. Binding sites of miR-619-5p in the coding regions of mRNAs of C8H8orf44, C8orf44, and ISY1 genes encode the WLMPVIP oligopeptide, which is present in the orthologous proteins. Binding sites of miR-619-5p in the mRNAs of transcription factor genes ZNF429 and ZNF429 encode the AHACNP oligopeptide in another reading frame. Binding sites of miR-619-5p in the 3’UTRs of all human target genes are also present in the 3’UTRs of orthologous genes of mammals. The completely complementary binding sites for miR-619-5p are conservative in the orthologous mammalian genes.ConclusionsThe majority of miR-619-5p binding sites are located in the 3’UTRs but some genes have miRNA binding sites in the 5’UTRs of mRNAs. Several genes have binding sites for miRNAs in the CDSs that are read in different open reading frames. Identical nucleotide sequences of binding sites encode different amino acids in different proteins. The binding sites of miR-619-5p in 3’UTRs, 5’UTRs and CDSs are conservative in the orthologous mammalian genes.

Molecular identification and phylogenetic analyses of multiple nucleopolyhedrovirus isolated from Lymantria obfuscata (Lepidoptera: Lymantriidae) in India

The Lymantria obfuscata Walker (Lyob) multiple (M) nucleopolyhedrovirus (NPV) (LyobMNPV) has been isolated and successfully applied for the management of the Indian gypsy moth, L. obfuscata in Jammu and Kashmir (J&K), India. The present work aimed to investigate the variability of LyobMNPV isolates from six localities of J&K through molecular [amplification of the polyhedrin (polh), late expression factor-8 (lef-8) and late expression factor-9 (lef-9) genes] and biological (bioassays) characterization. To identify the position of LyobMNPV in the phylogenetic tree of baculoviruses, partial sequences of the polh, lef-8 and lef-9 genes were determined by using the DNA sequences within their coding regions by optimizing the polymerase chain reaction with degenerate primers. The sequence alignment revealed that LyobMNPV isolates exhibited seven, five and eleven single nucleotide polymorphic sites in the case of polh, lef-8 and lef-9, respectively. The phylogenetic analyses supported placing LyobMNPV with the Lymantria dispar L. MNPV (LdMNPV) isolates from different countries, and showed that it was more closely related to LdMNPV than to Lymantria xylina Swinhoe NPV and Lymantria monacha L. NPV. The contaminated diet plug bioassays using 2nd instar larvae indicated that the median lethal dose (LD50) and median survival time (ST50) of different isolates of LyobMNPV against L. obfuscata were lower than those of LdMNPV against L. dispar. LyobMNPV was more closely related to LdMNPV but its LD50 and ST50 were lower than those of LdMNPV. The study provides novel information on the position of LyobMNPV in the phylogenetic tree of baculoviruses and about biological and genetic variation of Lymantria species’ NPV isolates from different parts of the world.

Y-family DNA polymerase-independent gap-filling translesion synthesis across aristolochic acid-derived adenine adducts in mouse cells

Translesion DNA synthesis (TLS) operates when replicative polymerases are blocked by DNA lesions. To investigate the mechanism of mammalian TLS, we employed a plasmid bearing a single 7-(deoxyadenosine-N6-yl)-aristolactam I (dA-AL-I) adduct, which is generated by the human carcinogen, aristolochic acid I, and genetically engineered mouse embryonic fibroblasts. This lesion induces A to T transversions at a high frequency. The simultaneous knockouts of the Polh, Poli and Polk genes did not influence the TLS efficiency or the coding property of dA-AL-I, indicating that an unknown DNA polymerase(s) can efficiently catalyze the insertion of a nucleotide opposite the adduct and subsequent extension. Similarly, knockout of the Rev1 gene did not significantly affect TLS. However, knockout of the Rev3l gene, coding for the catalytic subunit of polζ, drastically suppressed TLS and abolished dA-AL-I to T transversions. The results support the idea that Rev1 is not essential for the cellular TLS functions of polζ in mammalian cells. Furthermore, the frequency of dA-AL-I to T transversion was affected by a sequence context, suggesting that TLS, at least in part, contributes to the formation of mutational hot and cold spots observed in aristolochic acid-induced cancers.

An alphabaculovirus isolated from dead Lymantria dispar larvae shows high genetic similarity to baculovirus previously isolated from Lymantria monacha – An example of adaptation to a new host

A new isolate of baculovirus, Lymantria dispar multiple nucleopolyhedrovirus-BNP (LdMNPV-BNP), was found in dead gypsy moth (L. dispar) caterpillars collected in the Biebrzanski National Park in Poland. Here, we examined its biological activity, structure, genetic content and phylogeny. Multiple nucleocapsids of LdMNPV-BNP are enveloped together in 2–26 virions embedded in occluded bodies (OBs) very similar to the OBs previously described in viruses infecting Lymantriinae. This isolate kills pest larvae in a relatively short time (LT50 of approximately 9days for a dose of 2×107OBs/ml), highlighting the possibility for its use as a biopesticide. Next-generation sequencing of LdMNPV-BNP revealed gene content (e.g. DNA photolyase) that is not present in any LdMNPV isolate sequenced to date. The genome is 157,270 base pairs long and has a notably lower G+C content in comparison to other LdMNPVs (50.3% G+C content compared to an average of 57.4% among other LdMNPVs). According to our phylogenetic analysis based on 37 core genes, LdMNPV-BNP is a member of group II alphabaculoviruses, which are closely related to LdMNPV and LyxyMNPV (Lymantria xylina multiple nucleopolyhedrovirus). Molecular evolution inference based on the partial sequence of lef-8, lef-9 and polh genes shows that LdMNPV-BNP and isolates of Lymantria monacha nucleopolyhedrovirus (LymoNPV) may share a very recent common ancestor or be isolates of the same virus species. LdMNPV-BNP, like other baculoviruses, could be beneficial as an active component of biopesticides that can be used during forest integrated pest management.

Lymantria mathura nucleopolyhedrovirus: Identification, occurrence and genetic diversity in Iwate Prefecture, Japan

A high prevalence of nuclear polyhedrosis has been observed in larval outbreaks of Lymantria mathura in Iwate Prefecture, Japan. However, the virus responsible has not been identified. Here the virus was designated Lymantria mathura nucleopolyhedrovirus (LymaNPV), based on partial sequence results of the lef-8, lef-9, and polh genes and transmission electron microscopic observations. Diagnosis by polymerase chain reaction targeting of the partial polh gene and a subsequent restriction fragment length polymorphism (RFLP) analysis indicated that LymaNPV was an exclusive causative agent for the nuclear polyhedrosis of the L. mathura larvae. LymaNPV was also detected from a very small fraction of L. dispar larvae that co-occurred with L. mathura larvae where the prevalence of LymaNPV-caused disease was high. A bioassay using LymaNPV against L. dispar larvae produced infection with the inoculated virus. This finding was consistent with the results of the field sample analysis. LymaNPV contained six genome types in the L. mathura populations sampled, as determined by RFLP of LymaNPV genomic DNA and southern blot hybridization analyses. None of the genome types was unique to any sampling site, indicating that some flow had occurred among sites. However, genome-type composition seemed to differ among sites. This study provides basic information about the interaction between L. mathura and LymaNPV.

Management Of The Tent Caterpillar (Malacosoma indicum) Using Nucleopolyhedrovirus In Jammu & Kashmir, India

The tent caterpillar (Malacosoma indicum) is a serious pest that attacks a wide range of fruit and forest trees in Jammu & Kashmir (J & K) range of North-Western Himalayas. Investigations were carried out during the period March, 2012 to July, 2015 to isolate and bioprospect naturally occurring NPVs as potent biopesticides against this pest. The isolated viruses, M. indicum multiple nucleopolyhedrovirus (MainMNPV) were confirmed to be double stranded DNA virus belonging to the family Baculoviridae. Molecular identification of the viral DNA and determination of the partial sequences of the polh gene of MainMNPV isolates showed their similarity to the NPV of other insects and was more closely related to the M. californicum NPV in the NCBI taxonomy database. This is the first report from Indian subcontinent and worldwide about the occurrence of NPVs in natural larval populations of M. indicum. The field application of MainMNPV against the host populations on walnut with the pre-standardised dosage of 1 × 1012 POBs/acre reduced the larval population density up to 39-63%. In the year following application, the larval population density in the treated blocks was 24-28% less than in control blocks. Therefore, ecofriendly management of M. indicum is possible by the use of baculovirus based biopesticides in various cropping systems of J & K.

PP05.10 – 3086: Cockayne syndrome and DNA repair disorders: Novel expanding neurological phenotype

Objective Cockayne syndrome is an autosomal recessive leukodystrophy and multisystem disorder characterized by mental retardation, microcephaly, severe growth failure, sensorial impairment, peripheral neuropathy and cutaneous photosensitivity. It belongs to the family of DNA repair and transcription disorders together with xeroderma pigmentosum and trichothiodystrophy. Clinical and genetic diagnosis still remains challenging. Our goal was to develop a unique and efficient mutation-screening strategy for Cockayne syndrome and related disorders and to improve our understanding of the clinical spectrum of these conditions. Methods We have designed a novel targeted high-throughput sequencing approach for the diagnosis of Cockayne syndrome and related DNA repair disorders. We have studied 30 patients presenting with the cardinal clinical features of Cockayne syndrome or related DNA repair disorders. Results We have identified the causative mutations in 14 of the 30 patients. Three patients showed biallelic mutations in the ERCC6/CSB gene, 2 patients in the ERCC8/CSA gene, 1 patient in the ERCC2/XPD gene, 1 patient in the ERCC3/XPB, 1 patient in the ERCC5/XPG, 6 in the POLH gene. We present the clinical, neurological and radiological phenotypes of these patients with regard to our experience of a well-characterized cohort of more than 100 Cockayne patients previously studied in our center and we discuss the implications for diagnosis and prognosis purposes. Conclusion This novel approach enables us to rapidly confirm the genetic diagnosis of Cockayne syndrome and related disoders and to unravel the molecular defect in atypical and overlapping clinical presentations.

Remarkable induction of UV-signature mutations at the 3'-cytosine of dipyrimidine sites except at 5'-TCG-3' in the UVB-exposed skin epidermis of xeroderma pigmentosum variant model mice.

The human POLH gene is responsible for the variant form of xeroderma pigmentosum (XP-V), a genetic disease highly susceptible to cancer on sun-exposed skin areas, and encodes DNA polymerase η (polη), which is specialized for translesion DNA synthesis (TLS) of UV-induced DNA photolesions. We constructed polη-deficient mice transgenic with lacZ mutational reporter genes to study the effect of Polh null mutation (Polh(-/-)) on mutagenesis in the skin after UVB irradiation. UVB induced lacZ mutations with remarkably higher frequency in the Polh(-/-) epidermis and dermis than in the wild-type (Polh(+/+)) and heterozygote. DNA sequences of a hundred lacZ mutants isolated from the epidermis of four UVB-exposed Polh(-/-) mice were determined and compared with mutant sequences from irradiated Polh(+)(/)(+) mice. The spectra of the mutations in the two genotypes were both highly UV-specific and dominated by C→T transitions at dipyrimidines, namely UV-signature mutations. However, sequence preferences of the occurrence of UV-signature mutations were quite different between the two genotypes: the mutations occurred at a higher frequency preferentially at the 5'-TCG-3' sequence context than at the other dipyrimidine contexts in the Polh(+/+) epidermis, whereas the mutations were induced remarkably and exclusively at the 3'-cytosine of almost all dipyrimidine contexts with no preference for 5'-TCG-3' in the Polh(-/-) epidermis. In addition, in Polh(-/-) mice, a small but remarkable fraction of G→T transversions was also observed exclusively at the 3'-cytosine of dipyrimidine sites, strongly suggesting that these transversions resulted not from oxidative damage but from UV photolesions. These results would reflect the characteristics of the error-prone TLS functioning in the bypass of UV photolesions in the absence of polη, which would be mediated by mechanisms based on the two-step model of TLS. On the other hand, the deamination model would explain well the mutation spectrum in the Polh(+/+) genotype.

Site-directed Mutagenesis (Y52E) of POLH Affects Its Ability to Bypass Ultraviolet-induced DNA Lesions in HaCaT Cells.

DNA polymerase eta (Pol η) is one of several Y family trans-lesion synthesis (TLS) polymerases in humans and plays an important role in maintaining genome stability after ultraviolet (UV) irradiation, as it carries out error-free TLS at sites of UV-induced lesions. We performed site-directed mutagenesis of human polymerase η gene (Y52E), confirmed by sequencing, then cloned wild-type mutant and POLH genes into the eukaryotic vector pEGFP-N1. After transfecting wild-type and mutant plasmids into HaCaT keratinocytes, we tested for UV induced cis-syn cyclobutane pyrimidine dimer (CPDs) DNA lesions, and analysed cellular viability by MTT cell proliferation assay. The results showed that CPD levels decreased both with empty vector control (EVC), wild-type POLH, and Y52E-POLH over 48 hours post UV irradiation with 0.1 mW/cm2 UVB for 15 minutes (p = 0.025). The rate in CPD reduction of mutant POLH was less than in wild-type POLH. Cell viabilities of all three groups increased over 48 hours after UV irradiation, with the increased rate in the wild-type being higher than for mutant protein (p = 0.046). We conclude that Y52E POLH has reduced capacity to bypass UV induced DNA lesions in HaCaT cells.

Detrimental effects of UV‐B radiation in a xeroderma pigmentosum‐variant cell line

DNA polymerase η (pol η), of the Y-family, is well known for its in vitro DNA lesion bypass ability. The most well-characterized lesion bypassed by this polymerase is the cyclobutane pyrimidine dimer (CPD) caused by ultraviolet (UV) light. Historically, cellular and whole-animal models for this area of research have been conducted using UV-C (λ=100-280 nm) owing to its ability to generate large quantities of CPDs and also the more structurally distorting 6-4 photoproduct. Although UV-C is useful as a laboratory tool, exposure to these wavelengths is generally very low owing to being filtered by stratospheric ozone. We are interested in the more environmentally relevant wavelength range of UV-B (λ=280-315 nm) for its role in causing cytotoxicity and mutagenesis. We evaluated these endpoints in both a normal human fibroblast control line and a Xeroderma pigmentosum variant cell line in which the POLH gene contains a truncating point mutation, leading to a nonfunctional polymerase. We demonstrate that UV-B has similar but less striking effects compared to UV-C in both its cytotoxic and its mutagenic effects. Analysis of the mutation spectra after a single dose of UV-B shows that a majority of mutations can be attributed to mutagenic bypass of dipyrimidine sequences. However, we do note additional types of mutations with UV-B that are not previously reported after UV-C exposure. We speculate that these differences are attributed to a change in the spectra of photoproduct lesions rather than other lesions caused by oxidative stress.

Nonvirus encoded proteins could be embedded into Bombyx mori cypovirus polyhedra

To explore whether the nonvirus encoded protein could be embedded into Bombyx mori cypovirus (BmCPV) polyhedra. The stable transformants of BmN cells expressing a polyhedrin (Polh) gene of BmCPV were constructed by transfection with a non-transposon derived vector containing a polh gene. The polyhedra were purified from the midguts of BmCPV-infected silkworms and the transformed BmN cells, respectively. The proteins embedded into polyhedra were determined by mass spectrometry analysis. Host derived proteins were detected in the purified polyhedra. Analysis of structure and hydrophilicity of embedded proteins indicated that the hydrophilic proteins, in structure, were similar to the left-handed structure of polyhedrin or the N-terminal domain of BmCPV structural protein VP3, which were easily embedded into the BmCPV polyhedra. The lysate of polyhedra purified from the infected transformation of BmN cells with modified B. mori baculovirus BmPAK6 could infect BmN cells, indicating that B. mori baculovirus could be embedded into BmCPV polyhedra. Both the purified polyhedra and its lysate could be coloured by X-gal, indicating that the β-galactosidase expressed by BmPAK6 could be incorporated into BmCPV polyhedra. These results suggested that some heterologous proteins and baculovirus could be embedded into polyhedra in an unknown manner.

Classification, genetic variation and pathogenicity of Lymantria dispar nucleopolyhedrovirus isolates from Asia, Europe, and North America

Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) has been formulated and applied to control outbreaks of the gypsy moth, L. dispar. To classify and determine the degree of genetic variation among isolates of L. dispar NPVs from different parts of the range of the gypsy moth, partial sequences of the lef-8, lef-9, and polh genes were determined for Lymantria spp. virus samples from host populations throughout the world. Sequence analysis confirmed that all L. dispar virus samples tested contained isolates of the species Lymantria dispar multiple nucleopolyhedrovirus (Baculoviridae: Alphabaculovirus). Phylogenetic inference based on the lef-8 sequences indicated that the LdMNPV isolates formed two groups, one consisting primarily of isolates from Asia, and one consisting primarily of isolates from Europe and North America. The complete genome sequence was determined for an isolate from the Asian group, LdMNPV-2161 (S. Korea). The LdMNPV-2161 genome was 163,138bp in length, 2092bp larger than the previously determined genome of LdMNPV isolate 5–6 (CT, USA). The two genome sequences were co-linear, with an overall nucleotide sequence identity of 97.5% and some differences in ORF content. In droplet-feeding bioassays against neonate L. dispar larvae, isolates LdMNPV-3029 (Virin-ENSh/Russia) and LdMNPV-Ab-a624 (MA, USA) killed neonate larvae with an LC50 values that were 1.8- to 3.2-fold lower than a sample of Gypchek® (CT, USA) and isolates LdMNPV-3041 (Japan) and LdMNPV-2161. This study expands our knowledge about genetic variation among LdMNPV isolates and provides novel information on the distinct groups in which these NPVs occur.

Correlation of Phenotype/Genotype in a Cohort of 23 Xeroderma Pigmentosum-Variant Patients Reveals 12 New Disease-CausingPOLHMutations

Xeroderma pigmentosum variant (XP-V) is a rare genetic disease, characterized by some sunlight sensitivity and predisposition to cutaneous malignancies. We described clinical and genetic features of the largest collection ever published of 23 XPV patients (ages between 21 and 86) from 20 unrelated families. Primary fibroblasts from patients showed normal nucleotide excision repair but UV-hypersensitivity in the presence of caffeine, a signature of the XP-V syndrome. 87% of patients developed skin tumors with a median age of 21 for the first occurrence. The median numbers of basal-cell carcinoma was 13 per patient, six for squamous-cell carcinoma, and five for melanoma. XP-V is due to defects in the translesion-synthesis DNA polymerase Polη coded by the POLH gene. DNA sequencing of POLH revealed 29 mutations, where 12 have not been previously identified, leading to truncated polymerases in 69% of patients. Four missense mutations are correlated with the protein stability by structural modeling of the Polη polymerase domain. There is a clear relationship between the types of missense mutations and clinical severity. For truncating mutations, which lead to an absence of or to inactive proteins, the life-cumulated UV exposure is probably the best predictor of cancer incidence, reinforcing the necessity to protect XP-Vs from sun exposure.

Selection of a nucleopolyhedrovirus isolate fromHelicoverpa armigeraas the basis for a biological insecticide

The cotton bollworm, Helicoverpa armigera, is an insect that causes damage in a wide range of crops in Spain. Seven isolates of H. armigera single nucleopolyhedrovirus (HearSNPV) from the Iberian Peninsula were subjected to molecular and biological characterization and compared with a Chinese genotype (HearSNPV-G4). The estimated sizes of the Iberian genomes varied between 116.2 and 132.4 kb, compared to 131.4 kb of the HearSNPV-G4 reference genome. Phylogenetic analysis based on the lef-8, lef-9 and polh genes revealed that the Iberian strains were more closely related to one another than to other HearSNPV isolates. Occlusion body (OB) concentration-mortality responses (LC50 values) did not differ significantly among Iberian isolates when tested against a Helicoverpa armigera colony from Oxford (UK). Despite being the fastest killing isolate, HearSNPV-SP1 was as productive as isolates with lower virulence, with an average yield of 3.1 × 10(9) OBs larva(-1) . OBs of HearSNPV-SP1 and HearSNPV-G4 were similarly pathogenic against a recently established colony from southern Spain, although HearSNPV-SP1 was faster killing than HearSNPV-G4 against a range of instars. The insecticidal properties of HearSNPV-SP1 mean that this strain is likely to prove useful as the basis for a biological insecticide for control of Helicoverpa armigera in Spain.

A novel POLH mutation causes XP-V disease and XP-V tumor proneness may involve imbalance of numerous DNA polymerases

Xeroderma pigmentosum variant (XP-V) is a subtype of xeroderma pigmentosum (XP) disease with typical pigmentation and types of cancer in the oral maxillofacial and other sun-exposed regions. Few factors of tumor proneness in XP-V have been completely elucidated with the exception of the POLH [which encodes DNA polymerase η (pol η)] mutation. The aim of the present study was to identify the POLH mutation in an XP-V patient and to explore the roles of specific additional polymerases in XP-V tumor proneness. The POLH gene was sequenced in the patient and the expression of pol η, ι, κ, θ and ζ was tested in XP-V tumor cells and cell lines, as well as in HeLa cells with POLH knockdown. The results revealed a novel, large homozygous deletion of POLH (del exon 5–9) in the patient. Lower expression of pol κ, θ and ζ were observed in the XP-V cells and similar changes were observed in HeLa cells with POLH knockdown. Consistent with XP-V tumor cells, following UV irradiation, the expression of pol κ and θ presented was significantly increased in the XP-V cell lines compared with that in the normal control cells. The unusual expression of other polymerases, besides pol η, identified in the present study indicated that these polymerases may also be key in XP-V cells genetic instability, which accelerates tumor formation.