Haploadaptivity of tumor suppressor lgl and ontogenesis in Drosophila melanogaster: Increased survival rate and life span under stress conditions

Mutations of tumor suppressor lgl induce neuroblastoma and malignant transformation of epithelial larval tissues in Drosophila. We have already shown that heterozygotes for lethal null variants lgl/+ are widespread in natural populations. In order to elucidate this paradox, we analyzed the parameters of biological adaptation of the carriers of one dose of the tumor suppressor. We studied the patterns of embryonic survival rate of lgl/+ flies under the conditions of competition for life resources and development at elevated and lowered temperatures (29 and 16°C), influence of stress thermal conditions on longevity, influence of short-term temperature stress during prezygotic period in the course of oogenesis of mothers on survival rate of F1 progenies, and resistance of heterozygotes for different lethal lgl alleles against RNA virus DCV. The loss of one dose of tumor suppressor lgl provided for increased survival rate and longevity of lgl/+ heterozygotes under stress conditions. This phenomenon was called haploadaptivity. Important features of adaptogenesis were established in lgl/+ heterozygotes: dependence on the maternal genotype and critical periods in development. The increased survival rate of F1 progenies was determined already during early oogenesis of their lgl/+ mothers at the proembryo stage. With respect to humans, this conclusion draws attention to the oogenesis-dependent transgeneration aspect of determination and expression of mutant factors of risk, including tumor suppressors. The data obtained are essential for understanding of the causes of spreading null variants for the genes related to multiple pathologies, including cancer, in human populations.     

Elevated expression of p53 in early colon polyps in a pig model of human familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is a hereditary predisposition to formation of colon polyps that can progress to colorectal cancer (CRC). The severity of polyposis varies substantially within families bearing the same germline mutation in the adenomatous polyposis coli (APC) tumour suppressor gene. The progressive step-wise accumulation of genetic events in tumour suppressor genes and oncogenes leads to oncogenic transformation, with driver alterations in the tumour protein p53 (TP53) gene playing a key role in advanced stage CRC. We analysed groups of pigs carrying a truncating mutation in APC (APC^1311/+; orthologous to human APC^1309/+) to study the influence of TP53 polymorphisms and expression on the frequency of polyp formation and polyp progression in early-stage FAP. Five generations of APC^1311/+ pigs were examined by colonoscopy for polyposis severity and development. A total of 19 polymorphisms were found in 5′-flanking, coding, and 3′ untranslated regions of TP53. The distribution of TP53 genotypes did not differ between APC^1311/+ pigs with low (LP) and high (HP) number of colon polyps. p53 mRNA expression was analysed in distally located normal mucosa samples of wild-type pigs, APC^1311/+ LP and HP pigs, and also in distally located polyp samples histologically classified as low-grade (LG-IEN) and high-grade intraepithelial dysplastic (HG-IEN) from APC^1311/+ pigs. p53 mRNA expression was found to be significantly elevated in HG-IEN compared to LG-IEN samples (p?= 0.012), suggesting a role for p53 in the early precancerous stages of polyp development.     

Analysis of compound heterozygotes reveals that the mouse floxed <Emphasis Type="Italic">Pax6</Emphasis><Superscript><Emphasis Type="Italic">tm1Ued</Emphasis></Superscript> allele produces abnormal eye phenotypes

Analysis of abnormal phenotypes produced by different types of mutations has been crucial for our understanding of gene function. Some floxed alleles that retain a neomycin-resistance selection cassette (neo cassette) are not equivalent to wild-type alleles and provide useful experimental resources. Pax6 is an important developmental gene and the aim of this study was to determine whether the floxed Pax6 ^tm1Ued (Pax6 ^fl ) allele, which has a retained neo cassette, produced any abnormal eye phenotypes that would imply that it differs from the wild-type allele. Homozygous Pax6 ^fl/fl and heterozygous Pax6 ^fl/+ mice had no overt qualitative eye abnormalities but morphometric analysis showed that Pax6 ^fl/fl corneas tended be thicker and smaller in diameter. To aid identification of weak effects, we produced compound heterozygotes with the Pax6 ^Sey-Neu (Pax6 ^?) null allele. Pax6 ^fl/? compound heterozygotes had more severe eye abnormalities than Pax6 ^+/? heterozygotes, implying that Pax6 ^fl differs from the wild-type Pax6 ⁺ allele. Immunohistochemistry showed that the Pax6 ^fl/? corneal epithelium was positive for keratin 19 and negative for keratin 12, indicating that it was abnormally differentiated. This Pax6 ^fl allele provides a useful addition to the existing Pax6 allelic series and this study demonstrates the utility of using compound heterozygotes with null alleles to unmask cryptic effects of floxed alleles.     

Requirement of the isocitrate lyase gene <Emphasis Type="Italic">ICL1</Emphasis> for <Emphasis Type="Italic">VPS41</Emphasis>-mediated starvation response in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Cryptococcus neoformans is a major cause of fungal meningitis in individuals with impaired immunity. Our previous studies have shown that the VPS41 gene plays a critical role in the survival of Cryptococcus neoformans under nitrogen starvation; however, the molecular mechanisms underlying VPS41-mediated starvation response remain to be elucidated. In the present study, we show that, under nitrogen starvation, VPS41 strongly enhanced ICL1 expression in C. neoformans and that overexpression of ICL1 in the vps41 mutant dramatically suppressed its defects in starvation response due to the loss of VPS41 function. Moreover, targeted deletion of ICL1 resulted in a dramatic decline in viability of C. neoformans cells under nitrogen deprivation. Taken together, our data suggest a model in which VPS41 up-regulates ICL1 expression, directly or indirectly, to promote survival of C. neoformans under nitrogen starvation.     

Overexpression of <Emphasis Type="Italic">MeDREB1D</Emphasis> confers tolerance to both drought and cold stresses in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Cassava (Manihot esculenta) is an important tropical crop with extraordinary tolerance to drought stress but few reports on it. In this study, MeDREB1D was significantly and positively induced by drought stress. Two allelic variants of the gene named MeDREB1D(R-2) and MeDREB1D(Y-3) were identified. Overexpressing MeDREB1D(R-2) and MeDREB1D(Y-3) in Arabidopsis resulted in stronger tolerance to drought and cold stresses. Under drought stress, transgenic plants had more biomass, higher survival rates and less MDA content than wild-type plants. Under cold stress, transgenic plants also had higher survival rates than wild-type plants. To further characterize the molecular function of MeDREB1D, we conducted an RNA-Seq analysis of transgenic and wild-type Arabidopsis plants. The results showed that the Arabidopsis plants overexpressing MeDREB1D led to changes in downstream genes. Several POD genes, which may play a vital role in drought and cold tolerance, were up-regulated in transgenic plants. In brief, these results suggest that MeDREB1D can simultaneously improve plant tolerance to drought and cold stresses.     

Exocyst Complex Member EXOC5 Is Required for Survival of Hair Cells and Spiral Ganglion Neurons and Maintenance of Hearing

The exocyst, an octameric protein complex consisting of Exoc1 through Exoc8, was first determined to regulate exocytosis by targeting vesicles to the plasma membrane in yeast to mice. In addition to this fundamental role, the exocyst complex has been implicated in other cellular processes. In this study, we investigated the role of the exocyst in cochlear development and hearing by targeting EXOC5, a central exocyst component. Deleting Exoc5 in the otic epithelium with widely used Cre lines resulted in early lethality. Thus, we generated two different inner ear-specific Exoc5 knockout models by crossing Gfi1^Cre mice with Exoc5^f/f mice for hair cell-specific deletion (Gfi1^Cre/+;Exoc5^f/f) and by in utero delivery of rAAV-iCre into the otocyst of embryonic day 12.5 for deletion throughout the otic epithelium (rAAV2/1-iCre;Exoc5^f/f). Gfi1^Cre/+;Exoc5^f/f mice showed relatively normal hair cell morphology until postnatal day 20, after which hair cells underwent apoptosis accompanied by disorganization of stereociliary bundles, resulting in progressive hearing loss. rAAV2/1-iCre;Exoc5^f/f mice exhibited abnormal neurite morphology, followed by apoptotic degeneration of spiral ganglion neurons (SGNs) and hair cells, which led to profound and early-onset hearing loss. These results demonstrate that Exoc5 is essential for the normal development and survival of cochlear hair cells and SGNs, as well as the functional maintenance of hearing.     

Biogenesis of chlorophyll-binding proteins under iron stress in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

The biogenesis of chlorophyll-binding proteins under iron stress has been investigated in vivo in a chlN deletion mutant of Synechocystis sp. PCC 6803. The chlN gene encodes one subunit of the light-independent protochlorophyllide reductase. The mutant is unable to synthesize chlorophyll in darkness, causing chlorophyll biosynthesis to become light dependent. When the mutant was propagated in darkness, essentially no chlorophyll and photosystems were detected. Upon return of the chlN deletion mutant to light, 77 K fluorescence emission spectra and oxygen evolution of greening cells under iron-sufficient or-deficient conditions were measured. The gradual blue shift of the photosystem I (PS I) peak upon greening under iron stress suggested the structural alteration of newly synthesized PS I. Furthermore, the rate of biogenesis of PS II was delayed under iron stress, which might be due to the presence of IsiA.     

Heterozygosity for mutations affecting coat pigmentation in the American mink (<Emphasis Type="Italic">Neovison</Emphasis> vison) enhances structural stability of adrenal cortex under stress conditions

The results of the study of the effects of heterozygosity for mutations affecting coat pigmentation on the response to the environmental stress caused by extreme feeding conditions are provided. The animals with the following genotypes were taken into the study: homozygotes standard (+/+), hedlund white (h/h), and aleutian (a/a) and heterozygotes hedlund white (h/+) and aleutian (a/+). The animals homozygous for the aleutian mutation (a/a) showed a statistically lower growth rate than the animals of other genotypes both in the control and in the experiment (p < 0.05). Under the control conditions, the animals homozygous for both the wild type standard allele (+/+) and the mutant hedlund white (h/h) and aleutian (a/a) alleles showed the evident tendency for the zona fasciculata and zona reticularis of the adrenal cortex broadening compared to the experimental conditions. At the same time, in the animals heterozygous for the hedlund white (h/+) and the aleutian (a/+) mutations, a clear tendency for increasing size of the zona fasciculata and zona reticularis under the experimental conditions was observed. In the heterozygous animals, although we observed single destructive changes in the adrenal cortex under stress conditions, they were much less profound than in the homozygous ones. This may be related to the broader range of morphological adaptation in the heterozygotes, which gives them the possibility of more significant enlargement of the secreting zone to provide for its adequate functioning.     

Targeted Gene Deletion in <Emphasis Type="Italic">Cordyceps militaris</Emphasis> Using the Split-Marker Approach

The macrofungus Cordyceps militaris contains many kinds of bioactive ingredients that are regulated by functional genes, but the functions of many genes in C. militaris are still unknown. In this study, to improve the frequency of homologous integration, a genetic transformation system based on a split-marker approach was developed for the first time in C. militaris to knock out a gene encoding a terpenoid synthase (Tns). The linear and split-marker deletion cassettes were constructed and introduced into C. militaris protoplasts by PEG-mediated transformation. The transformation of split-marker fragments resulted in a higher efficiency of targeted gene disruption than the transformation of linear deletion cassettes did. The color phenotype of the Tns gene deletion mutants was different from that of wild-type C. militaris. Moreover, a PEG-mediated protoplast transformation system was established, and stable genetic transformants were obtained. This method of targeted gene deletion represents an important tool for investigating the role of C. militaris genes.     

Genes regulating the development and functioning of the nervous system determine life span in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Earlier, it has been shown that genes responsible for differences in longevity between wild-type Drosophila melanogaster lines 2b and Oregon are localized in region 7A6-B2, 36E4-37B9, 37B9-D2, and 64C-65C. Quantitative complementation tests were conducted between the gene mutations localized in these regions and involved in catecholamine biosynthesis (iav (inactive), Catsup (Catecholamines up), amd (alpha metil dopa-resistant), Dox-A2 (Diphenol oxidase A2), ple (pale)) and neuron development control (Fas3 (Fasciclin 3), tup (tail up), Lim3), on the one hand, and two different normal alleles of these genes in lines 2b and Oregon, on the other. Complementation was found for genes iav, Fas3, amd, and ple. The remaining genes (Catsup, Dox-A2, tup, and Lim3) are candidate genes for controlling differences in longevity between lines 2b and Oregon.     

The Effect of Mutation <Emphasis Type="Italic">dominant spotting-Yurlovo</Emphasis> (<Emphasis Type="Italic">Kit</Emphasis><Superscript><Emphasis Type="Italic">W-Y</Emphasis></Superscript>) on Spermatogenesis,Early Embryogenesis,and Fertility of C57BL/6JY Mice

The effect of mutation Kit ^W-Y found in C57BL/6 mice on fertility, spermatogenesis, and early embryogenesis of mice have been studied. If heterozygotes Kit ^W /+ are crossed with wild-type mice, fertility decreases by 20%. Homozygotes Kit ^W-Y /Kit ^W-Y and compounds Kit ^W-Y /Kit ^Ssm are nonviable. The study of spermatogenesis in Kit ^W /+ mice has demonstrated a negative effect of this mutation on spermatocytes. Histological examination of the testes of mutant males has shown local empty spaces in seminal ducts. Electron microscopic examination of synaptonemal complexes have demonstrated desynapsis disturbance in some nuclei at the diplotene stage of meiotic prophase I. However, these disturbances do not cause a decrease in the number of fertilized oocytes/ova. The decrease in fertility is accounted for disturbances of early embryogenesis. In vivo and in vitro analyses of early embryogenesis have demonstrated that cleavage divisions are asynchronous in Kit ^W-Y /+ heterozygous embryos. Some of these embryos die before implantation, and others cleave more rapidly than wild-type embryos, which give them selective advantage during the postimplantation period of embryogenesis. The pattern of Kit ^W-Y expression during spermatogenesis and embryogenesis mimics potential human pathology, which makes these mutants an interesting and valuable object for genetics and developmental biology.