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Specific-locus mutation assays in Zea mays. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

The use of the angiosperm Zea mays in assays for the induction of mutations at specific genetic loci is discussed. The effect of chemical mutagens on 3 genetic end points is evaluated. The end points surveyed were the loss of the phenotype of dominant alleles in heterozygotes, the induction of segregating point mutations in M2 plants, and the induction of forward and reverse mutations in pollen grains. Maize is sensitive to a wide range of mutagens and has the capacity to activate promutagens. This plant has been used to study mutagenesis under both laboratory and in situ conditions.

Genes↗

Weak androgen reduces the rate constant of beta-glucuronidase induction.

Administration of androgen to mice induces kidney beta-glucuronidase. Measuring beta-glucuronidase activity, rate of beta-glucuronidase synthesis, beta-glucuronidase mRNA activity and beta-glucuronidase mRNA concentration, the time course of induction was compared using a strong androgen, dihydrotestosterone (DHT), and a weakly androgenic progestin, medroxyprogesterone acetate (MPA). Using MPA resulted in a longer lag, a 3-4-fold slower rate of induction as defined by the forward rate constant, ka, a lower final extent of induction, and a slightly lower turnover constant, kb. Differences in kinetics of induction were consistent for all 4 measured parameters, and mimicked previously described genetic differences in these rate constants. The coordinate induction of beta-glucuronidase protein and beta-glucuronidase mRNA indicates that the response to androgen is regulated at a pre-translational level. That substitution of MPA for DHT decreases ka, rather than increasing kb, suggests that induction of beta-glucuronidase follows an increased rate of mRNA synthesis rather than a decreased rate of mRNA turnover. Finally, the results are consistent with a model in which the kinetic constants for beta-glucuronidase induction are dependent on the concentration of receptor molecules in the active conformational state.

Animals↗

Direct measurement of recombination frequency in interspecific hybrids between Hordeum vulgare and H. bulbosum using genomic in situ hybridization.

Two different genotypes of diploid Hordeum vulgare x H. bulbosum hybrids, which differ in their pattern of meiotic metaphase pairing behaviour, were investigated at MI and AI by genomic in situ hybridization (GISH). One hybrid, 102C2, showed a high frequency of bivalents at metaphase I whereas the other, 103K5, showed a high frequency of univalents. The GISH analysis of both hybrids established that pairing occurred only between chromosomes of different parental genomes and revealed that pairing frequency greatly exceeded recombination. Hybrid 102C2 had a significantly higher recombination frequency than 103K5, but in both hybrids recombination involved only distal chromosome regions. However, an interesting finding is that the ratio of recombination to pairing frequency in 103K5 (1:8.9) is twice as high compared with 102C2 (1:17). The hybrids also differed in chromosome stability; little chromosome elimination occurred in 102C2 but 103K5 showed extensive chromosome loss. It appears that the high frequency of bound arms at MI favours retention of H. bulbosum chromosomes and maintains stability of chromosome numbers in PMCs. Various ideas are put forward to explain the discrepancy between meiotic pairing frequency and recombination in these hybrids.

Hordeum↗

Topoisomerase II binds to ellipticine in the absence or presence of DNA. Characterization of enzyme-drug interactions by fluorescence spectroscopy.

Although a number of drugs currently in use for the treatment of human cancers act by stimulating topoisomerase II-mediated DNA breakage, little is known regarding interactions between these agents and the enzyme. To further define the mechanism of drug action, interactions between ellipticine (an intercalative drug with clinical relevance) and yeast topoisomerase II were characterized. By utilizing a yeast genetic system, topoisomerase II was identified as the primary cellular target of the drug. Furthermore, ellipticine did not inhibit enzyme-mediated DNA religation, suggesting that it stimulates DNA breakage by enhancing the forward rate of cleavage. Finally, ellipticine binding to DNA, topoisomerase II, and the enzyme-DNA complex was assessed by steady-state and frequency domain fluorescence spectroscopy. As determined by changes in fluorescence intensity and emission maximum wavelength, and by lifetime analysis, only the protonated species of ellipticine bound to a double-stranded 40-mer oligonucleotide containing a topoisomerase II cleavage site (KD approximately 65 nM). In contrast, predominantly deprotonated ellipticine bound to the enzyme.DNA complex (KD approximately 1.5 microM) or to the enzyme in the absence of nucleic acids (KD approximately 160 nM). These findings suggest that ellipticine interacts directly with topoisomerase II and that the enzyme dictates the ionic state of the drug in the ternary complex. A model is presented in which the topoisomerase II.ellipticine.DNA complex is formed via initial drug binding to either the enzyme or DNA.

Base Sequence↗

Hosts and parasites as aliens.

Over the past decades, various free-living animals (hosts) and their parasites have invaded recipient areas in which they had not previously occurred, thus gaining the status of aliens or exotics. In general this happened to a low extent for hundreds of years. With variable frequency, invasions have been followed by the dispersal and establishment of non-indigenous species, whether host or parasite. In the literature thus far, colonizations by both hosts and parasites have not been treated and reviewed together, although both are usually interwoven in various ways. As to those factors permitting invasive success and colonization strength, various hypotheses have been put forward depending on the scientific background of respective authors and on the conspicuousness of certain invasions. Researchers who have tried to analyse characteristic developmental patterns, the speed of dispersal or the degree of genetic divergence in populations of alien species have come to different conclusions. Among parasitologists, the applied aspects of parasite invasions, such as the negative effects on economically important hosts, have long been at the centre of interest. In this contribution, invasions by hosts as well as parasites are considered comparatively, revealing many similarities and a few differences. Two helminths, the liver fluke, Fasciola hepatica, of cattle and sheep and the swimbladder nematode, Anguillicola crassus, of eels are shown to be useful as model parasites for the study of animal invasions and environmental global change. Introductions of F. hepatica have been associated with imports of cattle or other grazing animals. In various target areas, susceptible lymnaeid snails serving as intermediate hosts were either naturally present and/or were introduced from the donor continent of the parasite (Europe) and/or from other regions which were not within the original range of the parasite, partly reflecting progressive stages of a global biota change. In several introduced areas, F. hepatica co-occurs with native or exotic populations of the congeneric F. gigantica, with thus far unknown implications. Over the fluke's extended range, in addition to domestic stock animals, wild native or naturalized mammals can also serve as final hosts. Indigenous and displaced populations of F. hepatica, however, have not yet been studied comparatively from an evolutionary perspective. A. crassus, from the Far East, has invaded three continents, without the previous naturalization of its natural host Anguilla japonica, by switching to the respective indigenous eel species. Local entomostrac crustaceans serve as susceptible intermediate hosts. The novel final hosts turned out to be naive in respect to the introduced nematode with far reaching consequences for the parasite's morphology (size), abundance and pathogenicity. Comparative infection experiments with Japanese and European eels yielded many differences in the hosts' immune defence, mirroring coevolution versus an abrupt host switch associated with the introduction of the helminth. In other associations of native hosts and invasive parasites, the elevated pathogenicity of the parasite seems to result from other deficiencies such as a lack of anti-parasitic behaviour of the naïve host compared to the donor host which displays distinct behavioural patterns, keeping the abundance of the parasite low. From the small amount of available literature, it can be concluded that the adaptation of certain populations of the novel host to the alien parasite takes several decades to a century or more. Summarizing all we know about hosts and parasites as aliens, tentative patterns and principles can be figured out, but individual case studies teach us that generalizations should be avoided.

Air Sacs↗

[A novel oligonucleotide arrays-based multiplex amplifiable probe hybridization technology].

Multiplex amplifiable probe hybridization (MAPH) has recently been developed to detect gene copy number changes in total genome in several genetic disease. Here we reported a novel oligonucleotide arrays-based multiplex amplifiable probe hybridization technology for DNA fragment copy number measurement. A set of amplifiable probes were prepared by locus-specific forward and reverse primers synthesized with the T7 and T3 promoter sites at their respective 5'-ends to the interest DNA fragments and purified by Qiagen PCR products purification kit. The set of probes were then hybridized with genome DNA immobilized on the nylon membrane. The selective probes after MAPH were collected with streptavidin coated magnetic beads and amplified by a biotin labeled universal primers. The biotinylated PCR products were then intended for hybridization to the corresponding oligonucleotide arrays consisted of 10 exons of DMD gene probes and positive,negative control probes immobilized on glass slides. After hybridization, the slides were washed by hybridization buffer and stained by streptavidin-Cy3. The hybridization fluorescence images were scanned by the ScanArray software and further quantitatively analyzed by the ImageJ software. Here we used oligonucleotide array technology to replace the agarose gel analysis for detecting the biotinylated PCR products so that extendable ability of parallel assay in our method had greatly been improved. One control female, one control male and one DMD patient were tested in this study. The results demonstrate the feasibility of high-throughput detection and relative quantification of DNA fragment copy numbers changes in total genome DNA combination of MAPH and high-density oligonucleotide arrays in single reaction.

Gene Dosage↗

Functional genomics of osteoarthritis: on the way to evaluate disease hypotheses.

Functional genomics is a challenging new way to address complex diseases such as osteoarthritis on a molecular level. This complements previous research and will open up new areas of so far unrecognized molecular networks. In this respect, articular cartilage is a good target for functional genomics as it contains only one cell type to which all expression signals can be attributed to. Despite considerable limitations at present, such as a low sensitivity and insensitivity to alternative splicing, posttranscriptional regulation, and posttranslational modification, cDNA-array technology provides a powerful tool to obtain an overview on gene expression patterns hardly achievable with other techniques. This has been shown to be true for known genes as well as for the identification of new genes of interest. Therefore, gene expression analysis will help to identify single genes depending on the disease and experimental conditions investigated. However, the expression pattern of the plethora of expressed genes will paint a picture (network) of disease context, maybe even more pushing forward our understanding of complex diseases such as osteoarthritis.

Cartilage, Articular↗

Multiple sclerosis. I. The immune pathogenetic hypothesis.

Animal models indicate that chronic relapsing demyelinating diseases of the central nervous system (CNS) can be induced both by certain viruses and by sensitization to myelinic antigens. Epidemiological data (studies on emigrants and on so-called "Multiple Sclerosis (MS) epidemics") suggest that an environmental transmissible agent, acting on a genetic substrate, could play a role in the pathogenesis of the disease. The transmissible agent could be a virus. Data to this effect are insufficiently indicative, but the hypothesis has been recently put forward that it could be antigenically related to the lymphotropic viruses (HTLV). The transmissible agent could act either by desegregating myelinic antigens or by inducing an alteration of the immune system with a reduction of T lymphocyte suppressor activity. There appears to be rapid and intense activated T lymphocyte "traffic" across the blood-brain barrier in patients affected with MS. Cells specifically sensitized to the unknown antigen find within the CNS other cells able to perpetuate presentation of the antigen (HLA-DR+ astrocytes and endothelial cells, not present in normal brain) and then activate the macrophages which in turn begin immuno-mediated phagocytosis of the healthy myelin. Aspecific immune cells are also recruited into CNS where seem to abnormally proliferate (possibly owing to reduced suppressor activity) producing antibodies against various viral or cerebral antigens which are not clearly related to the pathogenesis of the disease (production of intra-CNS "nonsense" antibodies and synthesis of the oligoclonal immunoglobulin bands seen at the cerebrospinal fluid electrophoresis.)

Antibodies, Monoclonal↗

Y chromosome and aggression in strains of laboratory mice.

Intermale attack behavior differences in laboratory strains of inbred mice have Y chromosome correlates in a small number of strain comparisons. Moreover, the Y correlates interact with autosomal or pseudoautosomal genes. Recent data on the genetics of the Y do not contradict these conclusions. The discovery of several polymorphic loci of the Y could pave the way for a direct confirmation of Y correlates of attack behavior by linkage detection. The involvement of the Y in this behavior has been put forward. Plasmatic testosterone concentration reactivity of some target organs to exogeneous testosterone appears to be correlated with two independent loci of the Y acting in an additive or interactive manner with autosomal or pseudoautosomal loci. However, the association between testosterone action and attack behavior in males needs further evidence, and in any case this association does not underline linear mechanisms.

Aggression↗

Pharmacogenetics of acute lymphoblastic leukemia.

PURPOSE OF REVIEW: The outcome in children with acute lymphoblastic leukemia has improved significantly over the past four decades. Current therapy results in event-free survival exceeding 80% for most patients. The development of risk-adapted therapy based on characteristics of the child (age), leukemia (leukocyte count, acquired genetic characteristics) and early response to therapy allows dose intensification for children with higher-risk disease. Much less attention has been given to the role of host variability (pharmacogenetic polymorphism) in determining outcome. This review discusses literature reports in this area and describes some of the challenges facing the field as it moves forward. RECENT FINDINGS: Polymorphisms in many different metabolic pathways have been demonstrated in single gene studies to influence the outcome of acute lymphoblastic leukemia. Challenges arise in establishing the generalizability of observations and interpreting complex gene-gene interactions in multigene pathways. Recent studies also illustrate the importance of correlation of clinical associations with biological mechanisms. SUMMARY: Despite significant progress in the treatment of childhood acute lymphoblastic leukemia, therapy is still unsuccessful in 20% of patients. Further knowledge of and insight into the role of host genetic polymorphisms will improve the results by integrating pharmacodynamic and pharmacogenomic studies in individualizing therapy for children with acute lymphoblastic leukemia.

Child↗

Health services implications of DNA testing.

This review article summarizes the state of the art in genetic testing and discusses the many issues that new technologies have raised. A health services perspective is offered to aid in providing laboratorians with an understanding of the dilemma that society faces with the exponential advances in knowledge. Unmistakably, these new technologies are a mixed blessing: on the one hand, diagnoses can be made with greater accuracy and preventive measures implemented more fruitfully and individuals may be more conclusively identified and/or exonerated for forensic purposes. On the other hand, however, are the very real concerns that discrimination under a medical guise will be encouraged and that privacy rights may be compromised. Another important issue is how the laboratory profession will serve in moving these new technologies from research to practice. We examine the role of the CLS in moving forward to a role of patient counselor and advocate in the emerging complex world of DNA-related biotechnology.

Bioethics↗

Peritoneal carcinoma in women with genetic susceptibility: implications for Jewish populations.

Women from families with multiple cases of breast and ovarian cancer, specifically those who carry cancer-associated mutations of BRCA1 or BRCA2 are at increased life-time risk for peritoneal carcinoma, even after previous surgery to remove the ovaries, fallopian tubes and uterus. Hereditary breast-ovarian cancer (HBOC) syndrome and the associated BRCA1 and BRCA2 mutations are particularly prevalent in women of Jewish lineage, and specific BRCA1 and BRCA2 germline mutations have been linked with peritoneal carcinoma and HBOC syndrome in Jewish populations, especially those of Ashkenazi descent. This review presents the currently available data and looks forward toward further and better understanding of peritoneal carcinoma in women with inherited susceptibility. Over 90% of peritoneal cancer in patients from HBOC syndrome kindreds and associated with BRCA1 and BRCA2 mutations are serous carcinomas, which is equivalent with the proportion of ovarian cancers that are serous carcinomas in similar patients. The best indications are that while many peritoneal carcinomas in genetically susceptible women may arise directly from malignant transformation of the peritoneum, others might represent metastases from primary ovarian or fallopian tube carcinomas. Although the incidence of borderline ovarian tumors may not be increased in HBOC syndrome kindreds and those who carry cancer-associated BRCA1 and BRCA2 mutations, these individuals could be susceptible to malignant transformation of borderline lesions of the ovaries and peritoneum. Moreover, recent reports raise the question of possibly increased risk in Jewish carriers of germline BRCA1 mutations for uterine papillary serous carcinoma, which could be the source of metastasis to the peritoneum in some cases. The penetrance of cancer-associated BRCA1 mutations for ovarian cancer is estimated to be 11%-54%, and for BRCA2 mutations the penetrance for ovarian cancer is 11%-23%. So far, available screening methods appear to be insufficient for early detection of many ovarian cancers. Prophylactic oophorectomy has been found to reduce the risk for ovarian cancer in women from HBOC kindreds and those who carry cancer-associated BRCA1 and BRCA2 mutations, leaving a residual risk for peritoneal carcinomatosis of well less than 5%. Therefore, surgical removal of the ovaries, fallopian tubes and uterus, after child-bearing has been completed and by early in the fifth decade of life, are appropriate prophylactic procedures in women whose genetic susceptibility puts them at increased risk for cancers of mullerian tract origin, including ovarian and fallopian tube carcinomas and possibly serous carcinoma of the uterus. Hysterectomy, as well as salpingo-oophorectomy, removes the gynecologic organs targeted for malignant transformation in genetically susceptible women and simplifies decisions regarding hormone replacement therapy and chemical prophylaxis and treatment of breast cancer. Unless a transabdominal operative approach is otherwise indicated, laparoscopic-assisted transvaginal techniques are well suited for intra-abdominal exploration, cytology, biopsies and prophylactic salpingo-oophorectomy and hysterectomy in women with hereditary susceptibility to gynecologic cancer.

Adult↗

Sweeps in Space: Leveraging Geographic Data to Identify Beneficial Alleles in Anopheles gambiae.

As organisms adapt to environmental changes, natural selection modifies the frequency of nonneutral alleles. For beneficial mutations, the outcome of this process may be a selective sweep, in which an allele rapidly increases in frequency and perhaps reaches fixation within a population. Selective sweeps have well-studied effects on patterns of local genetic variation in panmictic populations, but much less is known about the dynamics of sweeps in continuous space. In particular, because limited movement across a landscape leads to unique patterns of population structure, spatial dynamics may influence the trajectory of selected mutations. Here, we use forward-in-time, individual-based simulations in continuous space to study the impact of space on beneficial mutations as they sweep through a population. In particular, we show that selection changes the joint distribution of allele frequency and geographic range occupied by a focal allele and demonstrate that this signal can be used to identify selective sweeps. We then leverage this signal to identify in-progress selective sweeps within the malaria vector Anopheles gambiae, a species under strong selection pressure from vector control measures. By considering space, we identify multiple previously undescribed variants with potential phenotypic consequences, including mutations impacting known IR-associated genes and altering protein structure and properties. Our results demonstrate a novel signal for detecting selection in spatial population genetic data that may have implications for genomic surveillance and understanding geographic patterns of genetic variation.

Animals↗

Sweeps in space: leveraging geographic data to identify beneficial alleles in Anopheles gambiae.

As organisms adapt to environmental changes, natural selection modifies the frequency of non-neutral alleles. For beneficial mutations, the outcome of this process may be a selective sweep, in which an allele rapidly increases in frequency and perhaps reaches fixation within a population. Selective sweeps have well-studied effects on patterns of local genetic variation in panmictic populations, but much less is known about the dynamics of sweeps in continuous space. In particular, because limited movement across a landscape leads to unique patterns of population structure, spatial dynamics may influence the trajectory of selected mutations. Here, we use forward-in-time, individual-based simulations in continuous space to study the impact of space on beneficial mutations as they sweep through a population. In particular, we show that selection changes the joint distribution of allele frequency and geographic range occupied by a focal allele and demonstrate that this signal can be used to identify selective sweeps. We then leverage this signal to identify in-progress selective sweeps within the malaria vector Anopheles gambiae , a species under strong selection pressure from vector control measures. By considering space, we identify multiple previously undescribed variants with potential phenotypic consequences, including mutations impacting known IR-associated genes and altering protein structure and properties. Our results demonstrate a novel signal for detecting selection in spatial population genetic data that may have implications for genomic surveillance and understanding geographic patterns of genetic variation.

Journal Article↗

The nature of mutants induced by ionizing radiation in cultured hamster cells. II. Antigenic response and reverse mutation of HPRT-deficient mutants induced by gamma-rays or ethyl methanesulphonate.

A large series of independent mutants deficient in HPRT enzyme activity, isolated from V79-4 hamster cells (Brown and Thacker, 1984), were assessed for properties which reflect the nature of the genetic changes induced. A total of 88 mutants were screened, 43 isolated from gamma-ray-treated cultures and 45 induced by ethyl methanesulphonate (EMS). Firstly, each mutant was assayed for the presence of protein with the antigenic response of HPRT (cross-reacting material, CRM), using an antibody raised against partially purified V79-4 HPRT enzyme. In a competitive inhibition assay, 31% of EMS-induced mutants were CRM-positive compared to 7% of the gamma-ray series. Secondly, each mutant was tested for ability to revert to HPRT proficiency, either spontaneously or after treatment with the powerful mutagen ethyl nitrosourea (ENU). All except 2 of the EMS-induced mutants reverted with ENU, and many reverted spontaneously, under the given conditions. However reversion was not detected in about 80% of gamma-ray-induced mutants, suggesting that the types of forward mutation caused by ionizing radiation differ qualitatively from those caused by EMS. The EMS-induced mutations are likely to be mostly point mutations, with at least 40% of the missense type, while gamma-ray-induced mutations may arise mostly through larger genetic changes.

Animals↗

The segregation distortion (SD) phenomenon in wild populations of Drosophila melanogaster: interaction between chromosomes 3 and SD chromosomes 2.

The Segregation Distortion (SD) phenomenon is a typical case of non-Mendelian segregation in Drosophila melanogaster, due to the dysfunction of sperm bearing a non-SD homologous chromosome. In nature, several factors involved in the expression of the SD phenomenon have been described; among these, a genetic modifier carried by chromosome 3, which enhances the distortion effect of the SD chromosomes. The analysis of natural Sardinian populations, carried out in order to evaluate the presence of chromosome 3 bearing these enhancer factors, has enabled us to ascertain that (a) also in these populations chromosomes 3 with enhancer factors are present, although with frequencies lower than those previously reported in other publications; (b) among these enhancer chromosomes 3, some increase the k of certain chromosomes 2 from values of chromosomes considered non-distorting (k less than or equal to 0.66) to value typical of SD chromosomes. The data obtained also allow us to put forward some considerations regarding the dynamics of the SD phenomenon in Sardinian populations, where the frequency of SD chromosomes is fairly elevated.

Animals↗

Glucocorticoid secretion: a circadian synchronizer of the human temporal structure.

The circadian rhythm of glucocorticoid secretion represents an important endogenous synchronizer, a signal to place other bioperiodicities along an appropriate time scale. The results obtained both in experimental animals and in man by properly-timed administration of corticoids as well as personal observations in patients with Addison's disease given replacement therapy suggest that glucocorticoid action may not need continuous supply of hormone molecules to target cells but appropriate waves complying with the peripheral susceptibility. Furthermore, circadian changes in the susceptibility to glucocorticoid action may be phase-shifted among different targets. Evidence is presented that in human beings ACTH secretion is exquisitely sensitive to the negative feed-back effect by cortisol in the nocturnal hours following midnight, i.e. in a circadian stage preceding the acrophase of plasma glucocorticoid levels. In the circadian domain multiple rhythmic events, including chrono-feed-back and chrono-feed-forward mechanisms as well as the organization of steroid binding to plasma corticosteroid-binding globulin, provide stability, plasticity and economy to the activity of the entire hypothalamo-pituitary-adrenal system by conveying genetic and environmental control into a well-defined temporal program.

Addison Disease↗