The mouse mutants <Emphasis Type="Italic">recoil wobbler</Emphasis> and <Emphasis Type="Italic">nmf373</Emphasis> represent a series of <Emphasis Type="Italic">Grm1</Emphasis> mutations

The identification of novel mutant alleles is important for understanding critical functional domains of a protein and establishing genotype:phenotype correlations. The recoil wobbler (rcw) allelic series of spontaneous ataxic mutants and the ENU-induced mutant nmf373 genetically mapped to a shared region of chromosome 10. Their mutant phenotypes are strikingly similar; all have an ataxic phenotype that is recessive, early-onset, and is not associated with neurodegeneration. In this study we used complementation tests to show that these series of mutants are allelic to a knockout mutant of Grm1. Subsequently, a duplication of exon 4 and three missense mutations were identified in Grm1: I160T, E292D, and G337E. All mutations occurred within the ligand-binding region and changed conserved amino acids. In the rcw mutant, the Grm1 gene is expressed and the protein product is properly localized to the molecular layer of the cerebellar cortex. Grm1 is responsible for the generation of inositol 1,4,5-trisphosphate (IP₃). The inositol second messenger system is the central mechanism for calcium release from intracellular stores in cerebellar Purkinje cells. Several of the genes involved in this pathway are mutated in mouse ataxic disorders. The novel rcw mutants represent a resource that will have utility for further studies of inositol second-messenger-system defects in neurogenetic disorders.     

Interaction of gene <Emphasis Type="Italic">HSM3</Emphasis> with genes of the epistatic <Emphasis Type="Italic">RAD6</Emphasis> group in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

In eukaryotes, damage tolerance of matrix DNA is mainly determined by the repair pathway under the control of the RAD6 epistatic group of genes. This pathway is also a main source of mutations generated by mutagenic factors. The results of our recent studies show that gene HSM3 participating in the control of adaptive mutagenesis increases the frequency of mutations induced by different mutagens. Mutations rad18, rev3, and mms2 controlling various stages of the RAD6 pathway are epistatic with mutation hsm3 that decreases UV-induced mutagenesis to the level typical for single radiation-sensitive mutants. The level of mutagenesis in the double mutant srs2 hsm3 was lower than in both single mutants. Note that a decrease in the level of mutagenesis relative to the single mutant srs2 depends on the mismatch repair, since this level in the triple mutant srs2 hsm3 pms1 corresponds to that in the single mutant srs2. These data show that the mutator phenotype hsm3 is probably determined by processes occurring in a D loop. In a number of current works, the protein Hsm3 was shown to participate in the assembly of the proteasome complex S26. The assembly of proteasomes is governed by the N-terminal domain. Our results demonstrated that the Hsm3 protein contains at least two domains; the N-terminal part of the domain is responsible for the proteasome assembly, whereas the C-terminal portion of the protein is responsible for mutagenesis.     

A homozygous <Emphasis Type="Italic">RAB3GAP2</Emphasis> mutation causes Warburg Micro syndrome

Warburg Micro syndrome and Martsolf syndrome are clinically overlapping autosomal recessive conditions characterized by congenital cataracts, microphthalmia, postnatal microcephaly, and developmental delay. The neurodevelopmental and ophthalmological phenotype is more severe in Warburg Micro syndrome in which cerebral malformations and severe motor and mental retardation are common. While biallelic loss-of-function mutations in RAB3GAP1 are present in the majority of patients with Warburg Micro syndrome; a hypomorphic homozygous splicing mutation of RAB3GAP2 has been reported in a single family with Martsolf syndrome. Here, we report a novel homozygous RAB3GAP2 small in-frame deletion, c.499_507delTTCTACACT (p.Phe167_Thr169del) that causes Warburg Micro syndrome in a girl from a consanguineous Turkish family presenting with congenital cataracts, microphthalmia, absent visually evoked potentials, microcephaly, polymicrogyria, hypoplasia of the corpus callosum, and severe developmental delay. No RAB3GAP2 mutations were detected in ten additional unrelated patients with RAB3GAP1-negative Warburg Micro syndrome, consistent with further genetic heterogeneity. In conclusion, we provide evidence that RAB3GAP2 mutations are not specific to Martsolf syndrome. Rather, our findings suggest that loss-of-function mutations of RAB3GAP1 as well as functionally severe RAB3GAP2 mutations cause Warburg Micro syndrome while hypomorphic RAB3GAP2 mutations can result in the milder Martsolf phenotype. Thus, a phenotypic severity gradient may exist in the RAB3GAP-associated disease continuum (the “Warburg–Martsolf syndrome”) which is presumably determined by the mutant gene and the nature of the mutation.     

Possible use of <Emphasis Type="Italic">ail</Emphasis> and <Emphasis Type="Italic">foxA</Emphasis> polymorphisms for detecting pathogenic <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis>

Background  

Yersinia enterocolitica is an enteric pathogen that invades the intestinal mucosa and proliferates within the lymphoid follicles (Peyer's patches). The attachment invasion locus (ail) mediates invasion by Y. enterocolitica and confers an invasive phenotype upon non-invasive E. coli; ail is the primary virulence factor of Y. enterocolitica. The ferrioxamine receptor (foxA) located on the Y. enterocolitica chromosome, together with its transport protein, transports a siderophore specific for ferric ion. Currently, ail is the primary target gene for nucleic acid detection of pathogenic Y. enterocolitica.     

Effect of sporophytic <Emphasis Type="Italic">PIRL</Emphasis>9 genotype on post-meiotic expression of the Arabidopsis <Emphasis Type="Italic">pirl1</Emphasis>;<Emphasis Type="Italic">pirl9</Emphasis> mutant pollen phenotype

Plant intracellular ras-group-related leucine-rich repeat proteins (PIRLs) are a novel class of plant leucine-rich repeat (LRR) proteins structurally related to animal ras-group LRRs involved in cell signaling and gene regulation. Gene knockout analysis has shown that two members of the Arabidopsis thaliana PIRL gene family, PIRL1 and PIRL9, are redundant and essential for pollen development and viability: pirl1;pirl9 microspores produced by pirl1/PIRL1;pirl9 plants consistently abort just before pollen mitosis I. qrt1 tetrad analysis demonstrated that the genes become essential after meiosis, during anther stage 10. In this study, we characterized the phenotype of pirl1;pirl9 pollen produced by plants heterozygous for pirl9 (pirl1;pirl9/PIRL9). Alexander’s staining, scanning electron microscopy, and fluorescence microscopy indicated that pirl1;pirl9 double mutants produced by pirl9 heterozygotes have a less severe phenotype and more variable morphology than pirl1;pirl9 pollen from pirl1/PIRL1;pirl9 plants. Mutant pollen underwent developmental arrest with variable timing, often progressing beyond pollen mitosis I and arresting at the binucleate stage. Thus, although the pirl1 and pirl9 mutations act post-meiosis, the timing and expressivity of the pirl1;pirl9 pollen phenotype depends on the pirl9 genotype of the parent plant. These results suggest a continued requirement for PIRL1 and PIRL9 beyond the initiation of pollen mitosis. Furthermore, they reveal a modest but novel sporophytic effect in which parent plant genotype influences a mutant phenotype expressed in the haploid generation.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic variants of <Emphasis Type="Italic">CYP1B1</Emphasis> and <Emphasis Type="Italic">WDR36</Emphasis> in the patients with primary congenital glaucoma and primary open angle glaucoma from saint-Petersburg

In 32 unrelated patients with primary congenital glaucoma (PCG), a search for mutations in the myocilin (MYOC), cytochrome P450B1 (CYP1B1), and WDR36 genes was performed. The Q368X mutation in myocilin gene, typical of the patients with adult-onset primary open-angle glaucoma (POAG), was not detected in the PCG patients. Screening of the CYP1B1 introns 2 and 3 for the presence of mutations in PCG patients revealed only six DNA polymorphisms, including IVS1-12ntT>C (g.3793 T>C), A119S (g.4160 G>T; GCC>TCC), G188G (g.4369 C>A; GGC>GGA), L432V (g.8131 C>G; CTG>GTG), D449D (g.8184 C>T; GAC>GAT), and N453S (g.8195 A>G; AAC>AGC) (nucleotide numbering is given in accordance with the GenBank sequence U56438). In the groups of PCG patients and donors without eye diseases, the frequencies of these variants were not statistically significantly different, pointing to the neutrality of these polymorphisms. Furthermore, the CYP1B1 polymorphism L432V, considered to be associated with POAG in some world populations, was not associated with this disease in the patients from St. Petersburg. DNA collections obtained from the POAG and PCG patients and from the control group were tested for the carriage of the worldwide distributed mutations of the WRD36 gene, D658G, R529Q, A449T, and N355S. D658G variant was found with equally low frequencies in the groups of POAG and PCG patients, as well as in the control group. Mutations A449T and R529Q were found only once each, while mutation N355S was not detected in any of the groups examined. Our results indicate that the WDR36 variants make no substantial contribution to the development of POAG and PCG in the patients from St. Petersburg and represent normal DNA polymorphism. It is likely that in most of the PCG patients from the population examined the disease is not associated with the CYP1B1 gene defects.     

Mutations in the <Emphasis Type="Italic">S</Emphasis> gene region of hepatitis B virus genotype <Emphasis Type="Italic">D</Emphasis> in Turkish patients

The S gene region of the hepatitis B virus (HBV) is responsible for the expression of surface antigens and includes the ‘a’-determinant region. Thus, mutation(s) in this region would afford HBV variants a distinct survival advantage, permitting the mutant virus to escape from the immune system. The aim of this study was to search for mutations of the S gene region in different patient groups infected with genotype D variants of HBV, and to analyse the biological significance of these mutations. Moreover, we investigated S gene mutation inductance among family members. Forty HBV-DNA-positive patients were determined among 132 hepatitis B surface antigen (HbsAg) carriers by the first stage of seminested PCR. Genotypes and subtypes were established by sequencing of the amplified S gene regions. Variants were compared with original sequences of these serotypes, and mutations were identified. All variants were designated as genotype D and subtype ayw3. Ten kinds of point mutations were identified within the S region. The highest rates of mutation were found in chronic hepatitis patients and their family members. The amino acid mutations 125 (M → T) and 127 (T → P) were found on the first loop of ‘a’-determinant. The other consequence was mutation inductance in a family member. We found some mutations in the S gene region known to be stable and observed that some of these mutations affected S gene expression.     

<Emphasis Type="Italic">KRAS</Emphasis>, <Emphasis Type="Italic">BRAF</Emphasis>, <Emphasis Type="Italic">EGFR</Emphasis> and <Emphasis Type="Italic">HER2</Emphasis> gene status in a Spanish population of colorectal cancer

To evaluate the KRAS, BRAF, EGFR, and HER2 gene status in colorectal cancer by novel techniques and evaluate whether anti-HER2 therapies could be offered in the treatment of these patients. There are conflicting data on the prevalence of BRAF mutations and EGFR and HER2 gene amplification in colorectal KRAS wild type patients. In our study we tried to evaluate these expressions and their relationship to future treatment assays. Clinical–pathological data and paraffin-embedded specimens were collected from 186 patients who underwent colorectal resections at General Yagüe Hospital in Burgos, Spain. KRAS and BRAF status was analyzed by real-time PCR in all patients. EGFR and HER2/NEU gene amplification was detected using fluorescent in situ hybridisation technique (FISH) in 38 KRAS and BRAF wild type patients. KRAS mutations were present in 48% of the colorectal cancer patients. BRAF mutations were present in 6.25% of the KRAS wild type patients. EGFR and HER2 gene amplification was observed in 5.3% and 26.3%, respectively, of KRAS and BRAF wild type colorectal cancer patients. HER2, but not EGFR gene amplification, was frequently observed in KRAS and BRAF wild type colorectal cancer patients. These data indicate that HER2 amplification could be one of the genes to be considered in the therapeutic management of colorectal cancer.     

Functional conservation of the human <Emphasis Type="Italic">EXT1</Emphasis> tumor suppressor gene and its <Emphasis Type="Italic">Drosophila</Emphasis> homolog <Emphasis Type="Italic">tout</Emphasis><Emphasis Type="Italic">velu</Emphasis>

Heparan sulfate proteoglycans play a vital role in signaling of various growth factors in both Drosophila and vertebrates. In Drosophila, mutations in the tout velu (ttv) gene, a homolog of the mammalian EXT1 tumor suppressor gene, leads to abrogation of glycosaminoglycan (GAG) biosynthesis. This impairs distribution and signaling activities of various morphogens such as Hedgehog (Hh), Wingless (Wg), and Decapentaplegic (Dpp). Mutations in members of the exostosin (EXT) gene family lead to hereditary multiple exostosis in humans leading to bone outgrowths and tumors. In this study, we provide genetic and biochemical evidence that the human EXT1 (hEXT1) gene is conserved through species and can functionally complement the ttv mutation in Drosophila. The hEXT1 gene was able to rescue a ttv null mutant to adulthood and restore GAG biosynthesis.     

Overexpression of the <Emphasis Type="Italic">Activated Disease Resistance 1-like1</Emphasis> (<Emphasis Type="Italic">ADR1-L1</Emphasis>) Gene Results in a Dwarf Phenotype and Activation of Defense-Related Gene Expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The protein encoded by the activated disease resistance 1-like1 (ADR1-L1) gene (locus name, At4g33300) belongs to the activated disease resistance 1 (ADR1) family of coiled-coil nucleotide-binding site leucine-rich repeat-type disease resistance proteins. This family contains four proteins and they have specific features in their amino acid sequences. It has been reported that ADR1 protein belongs to the ADR1 family, which is related to not only defense response but also drought tolerance. We found that transgenic plants overexpressing the ADR1-L1 gene showed a dwarf phenotype and morphological change in leaves. The expression levels of defense-related genes and the resistance to Pseudomonas syringae pv. tomato DC3000 were increased in transgenic plants. However, enhancement of drought tolerance and activation of abiotic response genes were not observed. When the growth temperature was changed from 22°C to 28°C, the expression of defense-related genes and the enhancement of resistance to a bacterial pathogen were suppressed and the dwarf phenotype and morphological change of leaves recovered.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

<Emphasis Type="Italic">PIK3CA</Emphasis> mutations in <Emphasis Type="Italic">KRAS</Emphasis> and <Emphasis Type="Italic">BRAF</Emphasis> wild type colorectal cancer patients. A study of Spanish population

The objective of the work was to study PIK3CA mutations in wild type KRAS and BRAF colorectal cancer. Clinicopathological data and paraffin-embedded specimens were collected on 73 patients who underwent colorectal resections at General Yagüe Hospital in Burgos. KRAS, BRAF and PIK3CA status were analyzed by real-time PCR in all patients. PIK3CA mutations were present in 8.22% of wild type KRAS and BRAF colorectal cancers. The most frequent mutation is E545K/D in exon 9 which represents 83.3% of all mutations. By contrast, we did not found any tumour harbouring H1047R mutation in exon 20. Among the patients who undergo a curative resection of colorectal cancer, PIK3CA mutation is present in an important percentage of KRAS and BRAF wild type tumours. PIK3CA mutation may be considered as it could be a hypothetic reason to be not responder to anti-EGFR antibodies.     

Molecular evolution of the clustered <Emphasis Type="Italic">MIC</Emphasis>-<Emphasis Type="Italic">3</Emphasis> multigene family of <Emphasis Type="Italic">Gossypium</Emphasis> species

The Gossypium MIC-3 (Meloidogyne Induced Cotton-3) gene family is of great interest for molecular evolutionary studies because of its uniqueness to Gossypium species, multi-gene content, clustered localization, and root-knot nematode resistance-associated features. Molecular evolution of the MIC-3 gene family was studied in 15 tetraploid and diploid Gossypium genotypes that collectively represent seven phylogenetically distinct genomes. Synonymous (d_S) and non-synonymous (d_N) nucleotide substitution rates suggest that the second of the two exons of the MIC-3 genes has been under strong positive selection pressure, while the first exon has been under strong purifying selection to preserve function. Based on nucleotide substitution rates, we conclude that MIC-3 genes are evolving by a birth-and-death process and that a ‘gene amplification’ mechanism has helped to retain all duplicate copies, which best fits with the “bait and switch” model of R-gene evolution. The data indicate MIC-3 gene duplication events occurred at various rates, once per 1 million years (MY) in the allotetraploids, once per ~2 MY in the A/F genome clade, and once per ~8 MY in the D-genome clade. Variations in the MIC-3 gene family seem to reflect evolutionary selection for increased functional stability, while also expanding the capacity to develop novel “switch” pockets for responding to diverse pests and pathogens. Such evolutionary roles are congruent with the hypothesis that members of this unique resistance gene family provide fitness advantages in Gossypium.     

Increased tolerance to salt stress in the phosphate-accumulating <Emphasis Type="Italic">Arabidopsis</Emphasis> mutants <Emphasis Type="Italic">siz1</Emphasis> and <Emphasis Type="Italic">pho2</Emphasis>

High salinity is an environmental factor that inhibits plant growth and development, leading to large losses in crop yields. We report here that mutations in SIZ1 or PHO2, which cause more accumulation of phosphate compared with the wild type, enhance tolerance to salt stress. The siz1 and pho2 mutations reduce the uptake and accumulation of Na⁺. These mutations are also able to suppress the Na⁺ hypersensitivity of the sos3-1 mutant, and genetic analyses suggest that SIZ1 and SOS3 or PHO2 and SOS3 have an additive effect on the response to salt stress. Furthermore, the siz1 mutation cannot suppress the Li⁺ hypersensitivity of the sos3-1 mutant. These results indicate that the phosphate-accumulating mutants siz1 and pho2 reduce the uptake and accumulation of Na⁺, leading to enhanced salt tolerance, and that, genetically, SIZ1 and PHO2 are likely independent of SOS3-dependent salt signaling.     

Mutational analysis of the <Emphasis Type="Italic">ribC</Emphasis> gene of <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

The nucleotide sequence of the ribC gene encoding the synthesis of bifunctional flavokinase/flavine adenine nucleotide (FAD) synthetase in Bacillus subtilis have been determined in a family of riboflavinconstitutive mutants. Two mutations have been found in the proximal region of the gene, which controls the transferase (FAD synthase) activity. Three point mutations and one double mutation have been found (in addition to the two mutations that were detected earlier) in the distal region of the gene, which controls the flavokinase (flavin mononucleotide (FMN) synthase) activity. On the basis of all data known to date, it has been concluded that the identified mutations affect riboflavin and ATP binding sites. No mutations have been found in the PTAN conserved sequence, which forms the magnesium and ATP common binding site and is identical for organisms of all organizational levels, from bacteria too humans.     

Common <Emphasis Type="Italic">BRCA1</Emphasis> and <Emphasis Type="Italic">BRCA2</Emphasis> mutations in breast cancer families: a meta-analysis from systematic review

A number of molecular epidemiological studies have been conducted the screening for BRCA1 and BRCA2 mutations in breast cancer patients with a positive family history of breast and/or ovarian cancer and reported many common mutations in BRCA1 and BRCA2 associated in breast cancer in different population and different ethnicity. However, it’s still lack of a systematic analysis on these mutations. To comprehensively evaluate the frequency and distribution of common BRCA1 and BRCA2 mutations which associated with breast cancer risk, we address this issue through system review and meta-analysis on 29 relevant published studies by conducting a literature search on PubMed and CNKI. 20 common founder germline mutations were identified from all 29 studies and 4 of BRCA1 (5382insC, 185delAG, 3819del5 and 4153delA) and 2 of BRCA2 (4075delGT, 5802del4) mutations were repeatedly reported twice or more in different articles, respectively. For the BRCA1, after conducting meta-analysis, we found that the overall frequency of 5382insC was 0.09 (95% CI 0.06–0.12), the frequency of 185delAG was 0.07 (95% CI 0.01–0.13), the frequency of 3819del5 was 0.02 (95% CI 0.01–0.04) and the frequency of 4153delA was 0.06 (95% CI 0.03–0.09). For the BRCA2, the overall frequency of 4075delGT was 0.02 (95% CI 0.00–0.03) and the frequency of 5802del4 was 0.07 (95% CI 0.04–0.11). This article provides a set of common mutations for BRCA1 and BRCA2 mutation carriers and the results may help to explore frequencies of BRCA1 and BRCA2 mutations in a given population and will be of significance both for diagnostic testing and for epidemiological studies.     

Regulation of stipule development by <Emphasis Type="Italic">COCHLEATA</Emphasis> and <Emphasis Type="Italic">STIPULE</Emphasis>-<Emphasis Type="Italic">REDUCED</Emphasis> genes in pea <Emphasis Type="Italic">Pisum sativum</Emphasis>

Pisum sativum L., the garden pea crop plant, is serving as the unique model for genetic analyses of morphogenetic development of stipule, the lateral organ formed on either side of the junction of leafblade petiole and stem at nodes. The stipule reduced (st) and cochleata (coch) stipule mutations and afila (af), tendril-less (tl), multifoliate-pinna (mfp) and unifoliata-tendrilled acacia (uni-tac) leafblade mutations were variously combined and the recombinant genotypes were quantitatively phenotyped for stipule morphology at both vegetative and reproductive nodes. The observations suggest a role of master regulator to COCH in stipule development. COCH is essential for initiation, growth and development of stipule, represses the UNI-TAC, AF, TL and MFP led leafblade-like morphogenetic pathway for compound stipule and together with ST mediates the developmental pathway for peltate-shaped simple wild-type stipule. It is also shown that stipule is an autonomous lateral organ, like a leafblade and secondary inflorescence.