Association of MICA gene polymorphisms with liver fibrosis in schistosomiasis patients in the Dongting Lake region

Major histocompatibility complex class I chain-related A (MICA) is a highly polymorphic gene located within the MHC class I region of the human genome. Expressed as a cell surface glycoprotein, MICA modulates immune surveillance by binding to its cognate receptor on natural killer cells, NKG2D, and its genetic polymorphisms have been recently associated with susceptibility to some infectious diseases. We determined whether MICA polymorphisms were associated with the high rate of Schistosoma parasitic worm infection or severity of disease outcome in the Dongting Lake region of Hunan Province, China. Polymerase chain reaction-sequence specific priming (PCR-SSP) and sequencing-based typing (SBT) were applied for high-resolution allele typing of schistosomiasis cases (N = 103, age range = 36.2-80.5 years, 64 males and 39 females) and healthy controls (N = 141, age range = 28.6-73.3 years, 73 males and 68 females). Fourteen MICA alleles and five short-tandem repeat (STR) alleles were identified among the two populations. Three (MICA*012:01/02, MICA*017 and MICA*027) showed a higher frequency in healthy controls than in schistosomiasis patients, but the difference was not significantly correlated with susceptibility to S. japonicum infection (Pc > 0.05). In contrast, higher MICA*A5 allele frequency was significantly correlated with advanced liver fibrosis (Pc < 0.05). Furthermore, the distribution profile of MICA alleles in this Hunan Han population was significantly different from those published for Korean, Thai, American-Caucasian, and Afro-American populations (P < 0.01), but similar to other Han populations within China (P > 0.05). This study provides the initial evidence that MICA genetic polymorphisms may underlie the severity of liver fibrosis occurring in schistosomiasis patients from the Dongting Lake region.

MSX1 gene and nonsyndromic oral clefts in a Southern Brazilian population

Nonsyndromic oral clefts (NSOC) are the most common craniofacial birth defects in humans. The etiology of NSOC is complex, involving both genetic and environmental factors. Several genes that play a role in cellular proliferation, differentiation, and apoptosis have been associated with clefting. For example, variations in the homeobox gene family member MSX1, including a CA repeat located within its single intron, may play a role in clefting. The aim of this study was to investigate the association between MSX1CA repeat polymorphism and NSOC in a Southern Brazilian population using a case-parent triad design. We studied 182 nuclear families with NSOC recruited from the Hospital de Clínicas de Porto Alegre in Southern Brazil. The polymorphic region was amplified by the polymerase chain reaction and analyzed by using an automated sequencer. Among the 182 families studied, four different alleles were observed, at frequencies of 0.057 (175 bp), 0.169 (173 bp), 0.096 (171 bp) and 0.67 (169 bp). A transmission disequilibrium test with a family-based association test (FBAT) software program was used for analysis. FBAT analysis showed overtransmission of the 169 bp allele in NSOC (P=0.0005). These results suggest that the CA repeat polymorphism of theMSX1 gene may play a role in risk of NSOC in populations from Southern Brazil.

Single-nucleotide polymorphism analysis of GH, GHR, and IGF-1 genes in minipigs

Tibetan (TB) and Bama (BM) miniature pigs are two popular pig breeds that are used as experimental animals in China due to their small body size. Here, we analyzed single-nucleotide polymorphisms (SNPs) in gene fragments that are closely related to growth traits [growth hormone (GH), growth hormone receptor (GHR), and insulin-like growth factor (IGF)-1)] in these pig breeds and a large white (LW) control pig breed. On the basis of the analysis of 100 BMs, 108 TBs, and 50 LWs, the polymorphic distribution levels of GH, GHR, and IGF-1 were significantly different among these three pig breeds. According to correlation analyses between SNPs and five growth traits - body weight (BW), body length (BL), withers height (WH), chest circumference (CC), and abdomen circumference (AC) - three SNP loci in BMs and four SNP loci in TBs significantly affected growth traits. Three SNP sites in BMs and four SNP sites in TBs significantly affected growth traits. SNPs located in the GH gene fragment significantly affected BL and CC at locus 12 and BL at locus 45 in BMs, and also BW, WH, CC, and AC at locus 45 and WH and CC at locus 93 in TBs. One SNP at locus 85 in the BM GHR gene fragment significantly affected all growth traits. All indices were significantly reduced with a mixture of alleles at locus 85. These results provide more information regarding the genetic background of these minipig species and indicate useful selection markers for pig breeding programs.

Determination of CYP2D6 *3, *4, and *10 frequency in women with breast cancer in São Luís, Brazil, and its association with prognostic factors and disease-free survival

The CYP2D6 enzyme is crucial for the metabolism of tamoxifen. The CYP2D6 gene is highly polymorphic, and individuals can be extensive, intermediate, or poor tamoxifen metabolizers. The aim of this study was to determine the frequencies of the CYP2D6 *3, *4, and *10 alleles in women with breast cancer who were treated with tamoxifen and analyze the association of enzyme activity with prognostic factors and disease-free survival. We observed a high frequency of CYP2D6 *10, with an allelic frequency of 0.14 (14.4%). The *3 allele was not present in the studied population, and *4 had an allelic frequency of 0.13 (13.8%). We conclude that patients with reduced CYP2D6 activity did not present worse tumor characteristics or decreased disease-free survival than women with normal enzyme activity, as the difference was not statistically significant. We also observed a high frequency of CYP2D6 *10, which had not been previously described in this specific population. This study is the first in north-northeastern Brazil that aimed to contribute to the knowledge of the Brazilian regional profile for CYP2D6 polymorphisms and their phenotypes. These findings add to the knowledge of the distribution of different polymorphic CYP2D6 alleles and the potential role of CYP2D6 genotyping in clinical practice prior to choosing therapeutic protocols.

Haihdutusnopeuksien määritys glysiinin alipainekiteytyksessä

Alipainekiteytyksessä tasalaatuisen lopputuotteen valmistamiseksi kiteytysolosuhteiden vaikutus liuottimen haihtumisvuohon ja kiteiden ominaisuuksiin on huomioitava. Kandidaatin työn tavoitteena oli selvittää, miten glysiinin alipainekiteytyksessä vesiliuoksesta kiteytysolosuhteet (lämpötila ja paine) vaikuttavat liuottimen haihtumisvuohon ja syntyvien kiteiden polymorfiaan. Haihdutuskokeet suoritettiin puhtaalle vedelle ja alipainekiteytyskokeet glysiinin 95 % kylläiselle vesiliuokselle lämpötiloissa 40 °C, 50 °C ja 60 °C ja paineissa 40 mbar, 60 mbar ja 80 mbar. Kiteyttimenä käytettiin suljettua, virtaushaitoilla, sekoittimella ja ohjausputkella varustettua reaktoria johon oli kytketty alipainepumppu ja termostaatti paineen ja lämpötilan ohjaamiseksi. Nesteen määrä haihdutuskokeissa oli 2 kg ja alipainekiteytyskokeissa 1,2 kg. Osa alipainekiteytyskokeista saaduista kiteistä analysoitiin jauheröntgendiffraktioanalysaattorin (PXRD, engl. powder x-ray diffraction) avulla. Kaikki PXRD-laitteella analysoidut alipainekiteytyskokeissa saadut kiteet osoittautuivat glysiinin γ-polymorfiksi. Kiteytysolosuhteilla oli selkeä vaikutus haihtumisvuohon, mutta vaikutusta glysiinikiteiden polymorfiaan ei havaittu koealueella.

Sequence-characterized amplified region (SCAR) technique used for variety discrimination in vinca (Catharanthus roseus (L.) G.Don)

Sequence-Characterized Amplified Region (SCAR) appears as a useful technique for genetic purity testing and variety discrimination, applicable to species in which some other techniques have failed. In particular, this technique is very attractive with species in which RAPD results were not consistent. The RAPD polymorphic bands were cloned, sequenced and from the sequence information, primers pairs for normal PCR were developed. Since the probability of obtaining successful SCAR primers from RAPD polymorphic bands was about 50%, a larger number of RAPD polymorphic bands are needed to develop sufficient SCAR primers for varietal discrimination in vinca. In addition, the efficiency of the SCAR technique is strongly affected by the quality of DNA extracted from seeds. The SCAR banding patterns obtained from vinca seed were consistent and repeatable making the results reliable for genetic purity testing and variety discrimination. The SCAR technique is simple, fast, relatively inexpensive and allows the use of DNA extracted from dry seeds, which is very important in a seed-quality evaluating program

Genetic purity certificate in seeds of hybrid maize using molecular markers

One of the main features that confer high quality to the seed is its genetic purity, in which one of the major causes of contamination is the self-pollination of the female parent. Up to date, there is no accurate and fast methods for detecting such contamination. Thus, this work was carried out to certify the genetic purity in seeds of hybrid maize using different biochemical and DNA-based markers. Two single-cross hybrids and their parental lines derived from the maize breeding program at UFLA were evaluated by isoenzymatic pattern of alcohol dehydrogenase (ADH), esterase (EST), acid phosphatase (ACP), glutamate-oxaloacetate transaminase (GOT), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), phosphoglucomutase (PGM), 6-phosphoglucomate dehydrogenase (PGDH), catalase (CAT) and ß-glucosidade (ßGLU) and by microsatellites markers. The enzymatic systems that were able to distinguish the hybrids from their parental line were the catalase, the isocitrate dehydrogenase and the esterase. The esterase showed a Mendelian segregation pattern for UFLA 8/3 hybrid, that enables a safer genetic purity certificate. Microsatellites were able to differentiate the hybrid lines and the respective parental lines. Moreover, this technique was fast, precise and without environment effects. For microsatellites, the amplification pattern was identical when young leaves or seeds were used as DNA source. The possibility of using seeds as DNA source would accelerate and facilitate the role process of the genetic purity analysis.

Behaviour and life history of a large carpenter bee (Xylocopa virginica) in the northern extent of its range

Large carpenter bees (Hymenoptera: Apidae: Xylocopa) have traditionally been thought of as exhibiting solitary or occasionally communal colony social organization. However, studies have demonstrated more complex fonns of social behaviour in this genus. In this document, I examine elements ofbehaviour and life history in a North American species at the northern extreme of its range. Xylocopa virginica was found to be socially polymorphic with both solitary and meta-social or semi-social nests in the same population. In social nests, there is no apparent benefit from additional females which do not perfonn significant work or guarding. I found that the timing of life-history events varies between years, yet foraging effort only differed in the coldest and wettest year of2004 the study. Finally, I that male X virginica exhibit female defence polygyny, with resident and satellite males. Resident males maintain their territories through greater aggression relative to satellites.

Genetic variability in Larus argentatus and Sterna hirundo

Blood serum and egg-white protein samples from individuals representing seven colonies of Larusargentatus, and four colonies of Sterna hirundo were electrophoretically analysed to determine levels of genetic variability and to assess the utility of polymorphic loci as genetic markers. Variability occurred at five co-dominant autosomal loci. S. hirundo protein polymorphism occurred at the Est-5 and the Oest-l loci, while nineteen loci were monomorphic. L. argentatus samples were monomorphic at seventeen loci and polymorphic at the Ldh-A and the Alb loci. Intergeneric differences existed at the Oalb and the Ldh-A loci. Although LDH-A100 from both species possessed identical electrophoretLc mobilities, the intergeneric differences were expressed as a difference in enzyme the'ITIlostabilities. Geographical distribution of alleles and genetic divergence estimates suggest ~ hirundo population panmixis,at least at the sampled locations. The h argentatus gene pool appears relatively heterogeneous with a discreet Atlantic Coast population and a Great Lakes demic population. These observed population structures may be maintained by the relative amount of gene flow occurring within and among populations. Mass ringing data coupled to reproductive success information and analysis of dispersal trends appear to validate this assumption. Similar results may be generated by either selection or both small organism and low locus sample sizes. To clarify these results and to detect the major factor(s) affecting the surveyed portions of the genome, larger sample sizes in conjunction with precise eco-demographic data are required.

Mitochondrial inheritance in ustilago violacea

The anther smut fungus U stilago violacea has been developed as an important model organIsm for genetic, morphological and physiological studies. Valuable information on the nuclear genetics on U stilago violacea has been obtained in the last 20-25 years. However, in this organism almost nothing is known about mitochondria which make up an important aspect of the fungal genetic system. One fundamental aspect, mitochondrial inheritance, was addressed by this investigation. Mitochondrial DNA (mtDNA) of U. violacea was purified and restriction fragments cloned. MtDNA restriction fragment length polymorphisms (RFLPs) were identified among different isolates and were used as genetic markers for studying mitochondrial inheritance in crosses between polymorphic isolates. Matings of the yeast-like haploid cells of opposite mating types resulted in dikaryons containing mitochondria from both parents. The dikaryons were induced to form hyphae and then allowed to revert to haploid growth, resulting 1ll a colony that is bisectored for the two nuclear types. Both nuclear-type progeny of each cross were examined for parental mitochondrial type: Either mitochondrial type was observed 1ll the progeny. Thus, mitochondrial inheritance is biparental in this organism. The recovery of both mitochondrial types in the progeny was non-random. In progeny with the nuclear genotype of the al mating type parent mitochondria from both parents were inherited equally well. However, 1ll progeny with the a2 mating type, mitochondria were inherited almost exclusively (94%) from the a2 parent.

Intraspecific and interspecific social variation in the sweat bee Lasioglossum Malachurum and other members of the subgenus Evylaeus

Sweat bees exhibit a range of social behaviours, from solitary nesting, in which no workers are produced, to strong eusociality, in which workers exhibit a high degree of altruism, behaviour that is measured by the degree of personal reproductive sacrifice. Field studies were carried out for seven weeks during May-June 2000 in southern Greece in order to investigate intraspecific social variation, and test the hypothesis of a north-south cline of decreasing eusociality in the obligately eusocial sweat bee L. (E.) malachurum. A comparative study, using principal components analysis, was performed to determine if patterns of intraspecific social variation in L. malachurum reflect the patterns of social variation within the subgenus, Evylaeus, as a whole. The results of the field study reveal that, in Greece, two worker broods were produced followed by a third brood consisting of gynes, males and some workers, indicating that there was an overlap in worker and gyne production. There was strong caste distinction between queens and workers. Workers actively foraged and participated in nest construction as most workers (58%, n=303) had a high degree of mandibular wear. Workers did not participate in the oviposition of Brood 3 gynes since only 0.7% (n=278) of workers were mated. Furthermore, queen survival until the end of Brood 3 and a substantial size differential of 10.6% between queens and workers suggested that queen domination over worker behaviour during the early to mid-part of the colony cycle was plausible. Male production in Brood 3 by some workers was likely, since the timing of worker ovarian development corresponded with the timing of male production. These findings suggest that workers of the first two broods were primarily altruistic, but some (28%) Brood 1 (9%) and Brood 2 (19%) workers produced males, indicating that the degree of altruistic behaviour declined during the lifetime of the colony. In comparison with other L. malachurum populations in Europe, the Greek population of L. malachurum had a weaker social level as a result of the higher proportion of workers potentially involved in male production, thus 3 supporting the hypothesis of a southerly cline of decreasing eusociality. Furthermore, intraspecific variation in social level across Europe appears to be due to longer breeding seasons in more southerly locations that would promote the production of larger colonies and provide opportunities for workers to evade queen control. The comparative study using principal components analysis on 20 solitary (of the subgenera Evylaeus and Lasioglossum), eusocial and socially polymorphic Evylaeus species and populations reveals that six traits are closely associated with stronger eusociality in Evylaeus. These traits are: (1) a reduction in the proportion of males in the early brood(s); (2) a reduction in the proportion of females that mate; (3) an increase in the mean number of first brood workers; (4) a reduction in the proportion of females with developed ovaries; (5) an increase in size dimorphism between castes, and (6) nest guarding. These are traits that most significantly define principal component one and therefore distinguish social type as indicated by a clear separation of the eusocial and the solitary populations, with a socially polymorphic species falling in between. Furthermore, most of these traits are under foundress control and may suggest that the evolutionary loss or gain of eusociality is based on selection pressures on a founding female. Colony size and female ovarian development are common factors distinguishing social variation in L. malachurum and within the subgenus as a whole. The principal components analysis excluding the solitary species and the socially aberrant L. marginatum populations show the L. malachurum populations separated based on an increasing proportion of workers with developed ovaries as populations are found more south, lending further support to the hypothesis of a north-south cline of decreasing eusociality.

Cryptic species status of Anopheles (Diptera: Culicidae) mosquitoes in Canada using a multidisciplinary approach

Many species of Anopheles mosquitoes (Diptera: Culicidae) are now recognized as species complexes whose members are often indistinguishable morphologically but identifiable based on ecological, genetic, or behavioural data. Because the members of species complexes often differ in their vector potential, accurate identification of vector species is essential for successful mosquito control. To investigate the cryptic species status of Anopheles mosquitoes in Canada, specimens were collected from across the country and examined using morphological, molecular, and ecological data. Six of the seven traditionally recognised species from Canada were collected from locations in British Columbia, Quebec, Newfoundland and Labrador, and throughout Ontario, including Anopheles barberi, An. earlei, An. freeborni, An. punctipennis, An. quadrimaculatus s.l., and An. walkeri. Variation in polymorphic traits within An. earlei, An. punctipennis, and An. quadrimaculatus s.l. were quantified and egg morphology examined using scanning electron microscopy. Morphological identification of adult and larval specimens suggested that two described cryptic species, An. perplexens and An. smaragdinus, were present in Canada. DNA sequence data were analysed for evidence of cryptic species using three molecular markers: COl, ITS2, and ITS!. Intraspecific COl variation was very low in most species «1 %), except for An. punctipennis with 2% sequence divergence between those from British Columbia (BC) and Ontario (ON), and An. walkeri with 7% sequence divergence between populations from Manitoulin Island (NO) and Long Point Provincial Park (LP). Similar patterns were also seen using ITS2 and ITS 1. Therefore, molecular data revealed the presence of two putative cryptic species within two species examined (i.e., An. walkeri and An. punctipennis), corresponding to collection location (i.e., NO vs. LP and BC vs. ON, respectively). Surprisingly, there was no molecular support for the presence of either An. perplexens or An. smaragdinus in Canada despite the morphological assessments. Ecological data from all collection sites were recorded and are available in an online database designed to manage all collection and identification data. Current bionomic information, including regional abundance, larval habitat, and species associations, was determined for each species. This multidisciplinary study of Anopheles mosquitoes is the first detailed investigation of these potential disease vectors in Canada and demonstrates the importance of an integrated approach to anopheline systematics that includes molecular data.

Evolutionary Origin and Maintenance of Sociality in the Small Carpenter Bees

Many arthropods exhibit behaviours precursory to social life, including adult longevity, parental care, nest loyalty and mutual tolerance, yet there are few examples of social behaviour in this phylum. The small carpenter bees, genus Ceratina, provide important insights into the early stages of sociality. I described the biology and social behaviour of five facultatively social species which exhibit all of the preadaptations for successful group living, yet present ecological and behavioural characteristics that seemingly disfavour frequent colony formation. These species are socially polymorphic with both / solitary and social nests collected in sympatry. Social colonies consist of two adult females, one contributing both foraging and reproductive effort and the second which remains at the nest as a passive guard. Cooperative nesting provides no overt reproductive benefits over solitary nesting, although brood survival tends to be greater in social colonies. Three main theories explain cooperation among conspecifics: mutual benefit, kin selection and manipulation. Lifetime reproductive success calculations revealed that mutual benefit does not explain social behaviour in this group as social colonies have lower per capita life time reproductive success than solitary nests. Genetic pedigrees constructed from allozyme data indicate that kin selection might contribute to the maintenance of social nesting -, as social colonies consist of full sisters and thus some indirect fitness benefits are inherently bestowed on subordinate females as a result of remaining to help their dominant sister. These data suggest that the origin of sociality in ceratinines has principal costs and the great ecological success of highly eusociallineages occurred well after social origins. Ecological constraints such as resource limitation, unfavourable weather conditions and parasite pressure have long been considered some of the most important selective pressures for the evolution of sociality. I assessed the fitness consequences of these three ecological factors for reproductive success of solitary and social colonies and found that nest sites were not limiting, and the frequency of social nesting was consistent across brood rearing seasons. Local weather varied between seasons but was not correlated with reproductive success. Severe parasitism resulted in low reproductive success and total nest failure in solitary nests. Social colonies had higher reproductive success and were never extirpated by parasites. I suggest that social nesting represents a form of bet-hedging. The high frequency of solitary nests suggests that this is the optimal strategy when parasite pressure is low. However, social colonies have a selective advantage over solitary nesting females during periods of extreme parasite pressure. Finally, the small carpenter bees are recorded from all continents except Antarctica. I constructed the first molecular phylogeny of ceratinine bees based on four gene regions of selected species covering representatives from all continents and ecological regions. Maximum parsimony and Bayesian Inference tree topology and fossil dating support an African origin followed by an Old World invasion and New World radiation. All known Old World ceratinines form social colonies while New World species are largely solitary; thus geography and phylogenetic inertia are likely predictors of social evolution in this genus. This integrative approach not only describes the behaviour of several previously unknown or little-known Ceratina species, bu~ highlights the fact that this is an important, though previously unrecognized, model for studying evolutionary transitions from solitary to social behaviour.

Human Endogenous Retrovirus (HERV) Insertional Polymorphisms

Human endogenous retroviruses (HERVs) are the result of ancient germ cell infections of human germ cells by exogenous retroviruses. HERVs belong to the long terminal repeat (LTR) group of retrotransposons that comprise ~8% of the human genome. The majority of the HERVs documented have been truncated and/or incurred lethal mutations and no longer encode functional genes; however a very small number of HERVs seem to maintain functional in making new copies by retrotranspositon as suggested by the identification of a handful of polymorphic HERV insertions in human populations. The objectives of this study were to identify novel insertion of HERVs via analysis of personal genomic data and survey the polymorphism levels of new and known HERV insertions in the human genome. Specifically, this study involves the experimental validation of polymorphic HERV insertion candidates predicted by personal genome-based computation prediction and survey the polymorphism level within the human population based on a set of 30 diverse human DNA samples. Based on computational analysis of a limited number of personal genome sequences, PCR genotyping aided in the identification of 15 dimorphic, 2 trimorphic and 5 fixed full-length HERV-K insertions not previously investigated. These results suggest that the proliferation rate of HERVKs, perhaps also other ERVs, in the human genome may be much higher than we previously appreciated and the recently inserted HERVs exhibit a high level of instability. Throughout this study we have observed the frequent presence of additional forms of genotypes for these HERV insertions, and we propose for the first time the establishment of new genotype reporting nomenclature to reflect all possible combinations of the pre-integration site, solo-LTR and full-length HERV alleles.

Transposable Elements Are a Significant Contributor to Tandem Repeats in the Human Genome

Sequence repeats are an important phenomenon in the human genome, playing important roles in genomic alteration often with phenotypic consequences. The two major types of repeat elements in the human genome are tandem repeats (TRs) including microsatellites, minisatellites, and satellites and transposable elements (TEs). So far, very little has been known about the relationship between these two types of repeats. In this study, we identified TRs that are derived from TEs either based on sequence similarity or overlapping genomic positions. We then analyzed the distribution of these TRs among TE families/subfamilies. Our study shows that at least 7,276 TRs or 23% of all minisatellites/satellites is derived from TEs, contributing ∼0.32% of the human genome. TRs seem to be generated more likely from younger/more active TEs, and once initiated they are expanded with time via local duplication of the repeat units. The currently postulated mechanisms for origin of TRs can explain only 6% of all TE-derived TRs, indicating the presence of one or more yet to be identified mechanisms for the initiation of such repeats. Our result suggests that TEs are contributing to genome expansion and alteration not only by transposition but also by generating tandem repeats.