Search

Cadmium (Cd) and Mercury (Hg) is among the heavy metals most hazardous for plant and human health. Known to induce oxidative stress in plants and disbalance equilibrium in the antioxidant defence system, these metals alter plant growth and cause damage at the cellular and molecular levels. Soybean is an important oilseed crop that is raised in soils often contaminated by Cd and Hg. The comparative studies on the deleterious effect of Cd and Hg and the defence system of antioxidants were not studied earlier in soybean plant. In this study, soybean plants were exposed to Cd (100 ㎛ CdCl2) and Hg (100 ㎛ HgCl2) and studied for physiological, biochemical and molecular responses. Both Cd and Hg treatment increased the magnitude of oxidative stress. Activities of antioxidant enzymes were significantly upregulated in response to Cd and Hg stress. Quantitative and qualitative assessment of isolated RNA showed significant differences in RNA under stress. Integrity values of RNA confirmed alterations. Transcript level of the Actin gene, involved in the morphogenesis of plants and also used as referenced gene in expression studies was analyzed using qRT-PCR just to check its stability and response under heavy metal stress. Results showed significant upregulation of the gene in the presence of Cd. It can be concluded that both Cd and Hg caused oxidative damage to plants, and adversely affected the quality of RNA. However, soybean tried to limit the adverse impacts of Cd and Hg stress by elevating the antioxidant system and upregulating Actin gene. [ABSTRACT FROM AUTHOR]

Trichomoniasis is a common and curable sexually transmitted disease worldwide. The rapid, convenient, and accurate diagnosis of trichomoniasis is an important link in the prevention and treatment of the disease. The current detection methods of Trichomonas vaginalis are mainly wet mount microscopy, culture, nested PCR, and loop‐mediated isothermal amplification. However, these detection methods have some shortcomings. In this study, a recombinant enzyme polymerase amplification (RPA) assay had been conducted to detect T. vaginalis. The target gene and the corresponding primers were screened, and the reaction system and conditions were optimized in the assay of RPA. The sensitivity and specificity of this detection method were analyzed. The detection efficiency of wet mount microscopy, culture, nested PCR, and RPA was compared by testing 53 clinical samples from vaginal secretions. By screening, the actin gene of T. vaginalis could be used as a target gene for RPA detection of T. vaginalis, and the optimum reaction condition to amplify the actin gene by RPA was at 39°C for 30 min. The detection limit of T. vaginalis DNA using RPA was 1 pg, corresponding to a sensitivity of approximately five trophozoites. The RPA assay demonstrated high specificity for T. vaginalis, and there was no cross‐reactivity with Giardia lamblia, Escherichia coli, Lactobacillus, Toxoplasma gondii, Staphylococcus aureus, and Candida albicans. Of the 53 clinical samples, the positive rates of T. vaginalis detected by wet mount microscopy, culture, nested PCR and RPA were 50.9 4% (27/53), 71.7% (38/53), 71.7% (38/53), and 69.81% (37/53), respectively. Compared with culture which was used as the gold standard for diagnosing trichomoniasis, testing clinical samples by wet mount microscopy showed 71.05% sensitivity, 100% specificity, and moderate diagnostic agreement with the culture (K = 0.581, Z = 4.661, p < 0.001). The nested PCR showed 100% sensitivity, 100% specificity, and excellent diagnostic agreement (K = 1, Z = 7.28, p < 0.001), while RPA displayed 97.37% sensitivity, 100% specificity, and excellent diagnostic agreement (K = 0.954, Z = 6.956, p < 0.001). At the present study, rapid amplification of actin gene by RPA could be used as a tool for detection of T. vaginalis. The detection method of RPA was more sensitive than wet mount microscopy and displayed excellent specificity. Moreover, RPA amplification of actin gene did not require a PCR instrument and the amplification time was shorter than that of ordinary PCR. Therefore, the RPA assay was proposed in this study as a point‐of‐care examination and a diagnostic method of T. vaginalis infection, which exhibited the potential value in the treatment and prevention of trichomoniasis. [ABSTRACT FROM AUTHOR]

Carica papaya is a tropical fruit with immense health benefits and commercial importance. It is grown widely all over tropics and sub-tropics. Post-harvest pathogens are emerging as a major threat in the marketing and processing of papaya fruits. Soft rot symptom was beforehand established to be caused by fungal pathogen Rhizopus spp. The incidence of Gilbertella persicaria in papaya, producing similar symptoms have been identified in the fruits collected from college orchard of Tamil Nadu Agricultural University. Recently the fungus has being reported as causal organism of rot disease in papaya in few other countries. The responsible pathogen was identified as G. persicaria based on isolation, purification and further analysis. Morphological and molecular characterization was performed for pathogen identification. Molecular characterization was carried out using Internal Transcribed Spacer (ITS) region and Actin (Gil ACT) gene and phylogenetic analysis was done with concatenated sequence alignment in MEGA 11. Pathogenicity test was done using fresh papaya fruits to establish the pathogenic nature of the fungus and there by proved the Koch's postulate. • Gilbertella persicaria is an emerging pathogens affecting the fruit crops. • Role of the pathogen in inducing soft rot disease in post-harvest papaya is investigated. • Early detection and precautionary measures can overcome the possible yield loss under store house conditions in papaya. [ABSTRACT FROM AUTHOR]

Trichomonas vaginalis is a protozoan parasite that causes trichomoniasis with worldwide distribution. This study evaluated actin genotypes of T. vaginalis isolates using PCR–RFLP and sequence analysis in Tehran, Iran. Overall, 850 vaginal samples were collected from women admitted to hospitals affiliated with the Iran University of Medical Sciences in Tehran from 2020-to 2021. The samples were examined by wet mount and cultured. The parasites were harvested, and PCR–RFLP was performed using three endonuclease enzymes of HindII, MseI, and RsaI on all T. vaginalis isolates. Digestion patterns were then compared, and the genotype of these isolates was defined. The PCR products were sequenced. Overall, 12 (1.4%) isolates of T. vaginalis were identified from 850 vaginal samples collected. The most common genotypes were genotype E, seven (58.3%) and genotype G, three (25%), followed by genotype I, two (%16.7), using PCR–RFLP patterns and sequencing. No pattern indicative of mixed infection was found. PCR–RFLP is a proper technique to detect different T. vaginalis isolates, and noticeable polymorphism was found between isolates. Genotype E was the most common genotype in the studied group. The phylogenetic analysis indicated the T. vaginalis genotype E isolates in a distinct group compared to the genotypes G and I that evolved from a common ancestor. [ABSTRACT FROM AUTHOR]

Actin genes are genes that are common in organisms, and their expression is constitutive. These genes are used for gene normalization and internal control of DNA extraction, but the actin gene is not widely used for halal certification tests. Bioinformatic studies help to analyze the experiment through in silico more deeply before the experiment is carried out in laboratory, making it more efficient and time effective. uMelt is an analysis to predict the melting curve of target amplification in real-time PCR. Real-time PCR has been widely used for screening and detection of pork content in a product. This research aimed to explore actin gene as a candidate for testing pork using qPCR. The study was carried out in two main stages, namely alignment of the DNA sequence and analysis of the melting curve using the uMelt approach. The results showed a set of actin genes containing conserved regions that can be used as degenerate primers with different family-type coverages. Melting curve prediction with uMelt shows differences in tm peaks so as the types of samples can be easily identified. The use of bioinformatic applications such as uMelt helps in the simulation of predicting the melting curve to increase the precision of the analysis.

Here, we report the first case of Cryptosporidium cf. avium from an inland bearded dragon (Pogona vitticeps) from a wildlife sanctuary in Victoria, Australia. Molecular characterisation was conducted by PCR-coupled sequencing of regions in the small subunit of nuclear RNA (SSU), actin and large subunit of nuclear RNA (LSU) genes. The sequences obtained grouped with those of C. ornithophilus and other C. avium genotypes/variants originating from reptiles or birds. We discuss this case in relation to the current state of knowledge of C. avium of birds and reptiles, considering provenance and environment (agricultural, pet industry, wildlife, zoo or wildlife park) as well as clinical context, and pathological changes associated with cryptosporidiosis in these host animals.

Background: Trichomonas vaginalis (T. vaginalis) is a sexually transmitted protozoan parasite and the causative agent of trichomoniasis. The genetic characterization of T. vaginalis isolates shows notable genetic variation in this parasite. In the present study, we aimed to identify the T. vaginalis genotypes based on analyzing of actin gene in women specimens referred to health centers of Ilam city, southwest Iran. Methods: A total of 1765 female samples were collected from gynecology clinics in the city of Ilam. DNA was extracted from positive samples and nested polymerase chain reaction (Nested PCR) was used to amplify the actin gene. Then, partial sequencing and genotyping of the actin gene was performed. A phylogenetic tree was drawn using the detected genotypes of T. vaginalis and reference sequences. Results: Twenty-one of the 1765 urine and vaginal samples were positive for T. vaginalis. All infected individuals were married and their age in years was between 25 to 34. Further, the majority of infected women had cervical lesions, patchy erythema, and white color discharge. According to sequencing analysis, the isolates were identified as genotype G (n= 8) and genotype E (n= 2). Conclusions: From the collected samples, we were able to distinguish at least two genotypes (G and E) of T. vaginalis. However, lesser is known about these genotypes in the city of Ilam. Further studies with a higher number of isolates should be performed in order to understand the implications of these results in this region. [ABSTRACT FROM AUTHOR]

Here, we report the first case of Cryptosporidium cf. avium from an inland bearded dragon (Pogona vitticeps) from a wildlife sanctuary in Victoria, Australia. Molecular characterisation was conducted by PCR-coupled sequencing of regions in the small subunit of nuclear RNA (SSU), actin and large subunit of nuclear RNA (LSU) genes. The sequences obtained grouped with those of C. ornithophilus and other C. avium genotypes/variants originating from reptiles or birds. We discuss this case in relation to the current state of knowledge of C. avium of birds and reptiles, considering provenance and environment (agricultural, pet industry, wildlife, zoo or wildlife park) as well as clinical context, and pathological changes associated with cryptosporidiosis in these host animals. Image 1 • Characterisation of Cryptosporidium cf. avium from an inland bearded dragon (Pogona vitticeps). • Molecular differentiation of C. cf. avium from other members of the C. avium clade. • Pathogenicity of C. cf. avium seems higher than other members of this clade. [ABSTRACT FROM AUTHOR]

Abstract Background The protozoan parasite Trichomonas vaginalis is the most common non-viral, sexually transmitted pathogen. Although T. vaginalis is highly prevalent among women in Kenya, there is lack of data regarding genetic diversity of isolates currently in circulation in Kenya. Methods Typing was performed on 22 clinical isolates of T. vaginalis collected from women attending the antenatal care clinic at Kilifi County Hospital, Kenya, in 2015. Genotyping followed a previously proposed restriction fragment length polymorphism (RFLP) scheme, which involved in silico cleavage of the amplified actin gene by HindII, MseI and RsaI restriction enzymes. Phylogenetic analysis of all the sequences was performed to confirm the results obtained by RFLP-analysis and to assess the diversity within the RFLP genotypes. Additionally, we determined carriage of the four different types of Trichomonas vaginalis viruses (TVVs) by polymerase chain reaction. Results In silico RFLP-analysis revealed five actin genotypes; 50.0% of the isolates were of actin genotype E, 27.3% of actin genotype N, 13.6% of actin genotype G and 4.5% of actin genotypes I and P. Phylogenetic analysis was in agreement with the RFLP-analysis, with the different actin genotypes clustering together. Prevalence of TVVs was 43.5% (95% confidence interval, CI: 23.2–65.5). TVV1 was the most prevalent, present in 39.1% of the strains and 90% of the T. vaginalis isolates which harbored TVVs had more than one type of TVV. None of the isolates of actin genotype E harbored any TVV. Conclusion The presence of five actin genotypes in our study suggests notable diversity among T. vaginalis isolates occurring among pregnant women in Kilifi, Kenya. Isolates of the most prevalent actin genotype E lacked TVVs. We found no association between T. vaginalis genotype, carriage of TVVs and symptoms. Further studies with higher number of strains should be conducted in order to corroborate these results.

Objectives: C Trichomoniasis is one of the most common sexually transmitted diseases in the world, which is caused by Trichomonas vaginalis. It is also the most commonly reported sexually transmitted disease after the viral infections, which affects around 180 million people around the world each year. The people infected with this parasite exhibit a wide range of symptoms. To the best of our knowledge the genetic variation, prevalence and related factors affecting the disease have not been well studied. Therefore, this study aimed to determine the prevalence of T. vaginalis in women of southeast of Iran. Materials and Methods: Out of 500 patient women referred to the hospitals of Imam Khomeini in Zabol and Ali Ibn Abi Talib (AS) in Zahedan, 25 positive clinical samples were isolated from vaginal discharge and urine by culture method during June 2015 and May 2016. First, DNA was extracted and then all samples were subjected to nested PCR. Six different genotypes of actin gene were identified by PCR-RFLP in Trichomonas vaginalis in Zahedan and Zabol. All PCR products were digested with HindII, RsaI, and MesI restriction enzymes. All participants completed a questionnaire recommended by gynecologists and midwifery experts. Results: As a result, the genotypes of H, G, E, I, and N were identified in this study, from which the genotype E was the dominant genotype of T. vaginalis in Zahedan and Zabol. There was also a significant association between the type of clinical symptoms and the level of infection (P=0.0001). Conclusions: To sum up, disease as a health problem must be controlled through epidemiologic and genetic methods. Moreover, controlling the disease is closely associated with education and drug resistance or sensitivity related to genetic variation and epidemiologic factors. [ABSTRACT FROM AUTHOR]

Current knowledge of Cryptosporidium species/genotypes in marine fish is limited. Following phylogenetic analysis at the 18S rDNA locus, a recent study identified six new genotypes of Cryptosporidium colonizing edible fish found in European seas. Of these, five grouped in a clade together (#Cryptofish 1–5) and one grouped separately (#Cryptofish 7). In the present study, after phylogenetic analyses of #Cryptofish1, #Cryptofish2, #Cryptofish4, #Cryptofish5 and #Cryptofish7 at the actin locus, the presence of two major clades was confirmed. In addition, when possible, longer 18S amplicons were generated. In conclusion, the small genetic distances between these genotypes designated as a novel marine genotype I (#Cryptofish 1-5) suggest that they may be genetic variants of the same species, while the designated novel marine genotype 2 (#Cryptofish 7) is clearly representative of a separate species.

Actin is a multi-functional gene family that can be divided into muscle-type actins and non-muscle-type actins. In this study, 37 unigenes encoding actins were identified from RNA-Seq data of Pacific white shrimp, Litopenaeus vannamei. According to phylogenetic analysis, four and three cDNAs belong to cytoplasmic- and heart-type actins and were named LvActinCT and LvActinHT, respectively. 10 cDNAs belong to the slow-type skeletal muscle actins, and 18 belong to the fast-type skeletal muscle actins; they were designated LvActinSSK and LvActinFSK, respectively. Some muscle actin genes formed gene clusters in the genome. Multiple alternative transcription starts sites (ATSSs) were found for LvActinCT1. Based on the early developmental expression profile, almost all LvActins were highly expressed between the early limb bud and post-larval stages. Using LvActinSSK5 as probes, slow-type muscle was localized in pleopod muscle and superficial ventral muscle. We also found three actin genes that were down-regulated in the hemocytes of white spot syndrome virus (WSSV)- and Vibrio parahaemolyticus-infected L. vannamei. This study provides valuable information on the actin gene structure of shrimp, furthers our understanding of the shrimp muscle system and helps us develop strategies for disease control and sustainable shrimp farming. [ABSTRACT FROM AUTHOR]

Background: The present study aimed to characterize genetically and to compare the most frequently occurring strains of Trichomonas vaginalis isolated from southern Iran.Methods: Totally, 150 vaginal swab and urine specimens were collected from symptomatic and asymptomatic women from May 2012 to Jun 2013.This study implemented a sensitive and reliable PCR-restriction fragment length polymorphism (RFLP) typing method on the actin gene. Moreover, one representative sample of each identified genotype was subjected to sequencing.Results: Twenty-four T. vaginalis isolates were positive and6 distinct electrophoretic patterns (H, E, G, I, M, N) were identified. Genotypes H and I were found to be more prevalent (50 and 37.5%) in Kerman and Shiraz, respectively. The phylogenetic analysis showed that two isolates were located as a separated clade with the other T. vaginalis isolates.Conclusion: The obtained findings showed a considerable genetic polymorphism of clinical isolates from the population studied. More studies may be warranted in future as to unveiling any possible links between a given genotype/cluster and pathogenic behavior of T. vaginalis.

In animals and land plants, many asexual species originate through inter- or intraspecific crosses, and such heterozygous asexuals frequently are more abundant than their sexual relatives in marginal habitats. Although asexual species have been reported in various macroalgal taxa, detailed information regarding their distribution, heterozygosity, and origin is limited. Because many asexual tetrasporophyte strains of Caloglossa vieillardii have been isolated from South Australia, far from their core tropical habitats, we re-examined the distribution range of asexual C. vieillardii and genotyped these and other western Pacific strains using an actin gene marker. We confirmed the marginal distribution of the asexuals; however, a small patch of sexual thalli was newly discovered 450 km further west from asexual populations in South Australia. Three heterozygous genotypes and one homozygous genotypes were detected from nine asexual populations; 21 heterozygous strains were obligately asexual, but one homozygous strain suddenly produced sexual gametophytes after several years of culture. We hypothesized that the most abundant heterozygous genotype (defined as type 3/4) in asexual populations occurred by a cross between type 3 and type 4 allele gametophytes, both of which were isolated from the Australian coasts. In the crossing experiments, certain combinations between type 3 females and type 4 males produced tetrasporophytes, which recycled successive tetrasporophytes. In the culture experiments, whereas both sexual and asexual strains successfully produced tetraspores at 12°C, no sexual strains released carpospores below 14°C. However, it is uncertain whether this slight difference of maturation temperature was related to the marginal distribution of asexual C. vieillardii. [ABSTRACT FROM AUTHOR]

Currently, the systematics, biology and epidemiology of piscine Cryptosporidium species are poorly understood. Here, we compared Sanger ‒ and next-generation ‒ sequencing (NGS), of piscine Cryptosporidium, at the 18S rRNA and actin genes. The hosts comprised 11 ornamental fish species, spanning four orders and eight families. The objectives were: to (i) confirm the rich genetic diversity of the parasite and the high frequency of mixed infections; and (ii) explore the potential of NGS in the presence of complex genetic mixtures. By Sanger sequencing, four main genotypes were obtained at the actin locus, while for the 18S locus, seven genotypes were identified. At both loci, NGS revealed frequent mixed infections, consisting of one highly dominant variant plus substantially rarer genotypes. Both sequencing methods detected novel Cryptosporidium genotypes at both loci, including a novel and highly abundant actin genotype that was identified by both Sanger sequencing and NGS. Importantly, this genotype accounted for 68·9% of all NGS reads from all samples (249 585/362 372). The present study confirms that aquarium fish can harbour a large and unexplored Cryptosporidium genetic diversity. Although commonly used in molecular parasitology studies, nested PCR prevents quantitative comparisons and thwarts the advantages of NGS, when this latter approach is used to investigate multiple infections. [ABSTRACT FROM PUBLISHER]

Background: The protozoan parasite Trichomonas vaginalis is the most common non-viral, sexually transmitted pathogen. Although T. vaginalis is highly prevalent among women in Kenya, there is lack of data regarding genetic diversity of isolates currently in circulation in Kenya. Methods: Typing was performed on 22 clinical isolates of T. vaginalis collected from women attending the antenatal care clinic at Kilifi County Hospital, Kenya, in 2015. Genotyping followed a previously proposed restriction fragment length polymorphism (RFLP) scheme, which involved in silico cleavage of the amplified actin gene by HindII, MseI and RsaI restriction enzymes. Phylogenetic analysis of all the sequences was performed to confirm the results obtained by RFLP-analysis and to assess the diversity within the RFLP genotypes. Additionally, we determined carriage of the four different types of Trichomonas vaginalis viruses (TVVs) by polymerase chain reaction. Results: In silico RFLP-analysis revealed five actin genotypes; 50.0% of the isolates were of actin genotype E, 27.3% of actin genotype N, 13.6% of actin genotype G and 4.5% of actin genotypes I and P. Phylogenetic analysis was in agreement with the RFLP-analysis, with the different actin genotypes clustering together. Prevalence of TVVs was 43.5% (95% confidence interval, CI: 23.2-65.5). TVV1 was the most prevalent, present in 39.1% of the strains and 90% of the T. vaginalis isolates which harbored TVVs had more than one type of TVV. None of the isolates of actin genotype E harbored any TVV. Conclusion: The presence of five actin genotypes in our study suggests notable diversity among T. vaginalis isolates occurring among pregnant women in Kilifi, Kenya. Isolates of the most prevalent actin genotype E lacked TVVs. We found no association between T. vaginalis genotype, carriage of TVVs and symptoms. Further studies with higher number of strains should be conducted in order to corroborate these results. [ABSTRACT FROM AUTHOR]

Background: The present study aimed to characterize genetically and to compare the most frequently occurring strains of Trichomonas vaginalis isolated from southern Iran. Methods: Totally, 150 vaginal swab and urine specimens were collected from symptomatic and asymptomatic women from May 2012 to Jun 2013.This study implemented a sensitive and reliable PCR-restriction fragment length polymorphism (RFLP) typing method on the actin gene. Moreover, one representative sample of each identified genotype was subjected to sequencing. Results: Twenty-four T. vaginalis isolates were positive and6 distinct electrophoretic patterns (H, E, G, I, M, N) were identified. Genotypes H and I were found to be more prevalent (50 and 37.5%) in Kerman and Shiraz, respectively. The phylogenetic analysis showed that two isolates were located as a separated clade with the other T. vaginalis isolates. Conclusion: The obtained findings showed a considerable genetic polymorphism of clinical isolates from the population studied. More studies may be warranted in future as to unveiling any possible links between a given genotype/cluster and pathogenic behavior of T. vaginalis. [ABSTRACT FROM AUTHOR]

Uninucleated Entamoeba cysts measuring 7.3 × 7.7 μm were detected in faecal samples collected from wild Rangeland goats ( Capra hircus ) after arrival at a commercial goat depot near Geraldton, Western Australia at a prevalence of 6.4% (8/125). Sequences were obtained at the 18S rRNA (n = 8) and actin (n = 5) loci following PCR amplification. At the 18S locus, phylogenetic analysis grouped the isolates closest with an E. bovis isolate (FN666250) from a sheep from Sweden with 99% similarity. At the actin locus, no E. bovis sequences were available, and the isolates shared 94.0% genetic similarity with E. suis from a pig in Western Japan. This is the first report to describe the morphology and molecular characterisation of Entamoeba from Rangeland goats in Western Australia and the first study to produce actin sequences from E. bovis -like Entamoeba sp. [ABSTRACT FROM AUTHOR]

Background: The aim of this study was to identify the Trichomonas vaginalis strains/haplotypes based on identifying their probable variations in asymptomatic patients referred to Tabriz health centers, northwestern Iran. Methods: Sampling was taken from 50-suspected women to T. vaginalis in northwestern Iran. The obtained samples were smeared and cultured. Fifty DNA samples were extracted, amplified and identified by nested polymerase chain reaction and PCR-RFLP of actin gene using two endonuclease enzymes: MseI and RsaI. To reconfirm, the amplicons of actin gene were directly sequenced in order to identify the strains/haplotypes. Results: PCR-RFLP patterns, sequencing and phylogenetic analyses revealed definitely the presence of the G (n=22; 73.4%) and E (n=8; 26.6%) strains. Multiple alignments findings of genotype G showed five haplotypes and two amino acid substitutions in codons 192 and 211 although, no remarkable unique haplotype was found in genotype E. Conclusion: The accurate identification of T. vaginalis strains based on discrimination of their unknown haplotypes particularly those which are impacted on protein translation should be considered in parasite status, drug resistance, mixed infection with HIV and monitoring of asymptomatic trichomoniasis in the region.

An alpha actin gene segment, isolated from Nile tilapia (Oreochromis niloticus), was characterized by nucleotide sequencing, predicted amino acid sequence and Southern blot hybridization. Genomic DNA amplification resulted in a 1063-bp fragment corresponding to a partial alpha-cardiac muscle actin gene containing exons 3 to 6. Southern blot analysis of the restriction-digested DNA revealed that the Nile tilapia genome contains multiple muscle actin isoforms. Although comparison of the nucleotide sequence, amino acid residues and exon-intron organization of the isolated actin gene with those of other vertebrates showed a high level of identity, diagnostic amino acid residues can still be correlated to distinct actin genes in fish species.

For consumers, honey is a natural product that should not be subjected to treatment or alteration. Since the question of the presence of genetically modified organisms concerned pollen in honey, the aim of this research was to find an alternative, however, practical and efficient method of honey and bee pollen DNA extraction for routine analysis application. Furthermore, to evaluate the extracted DNA, a real-time PCR system based on the actin gene was optimized and validated in fast mode for the first time to reduce analysis time. To develop an alternative DNA extraction protocol, two already published procedures were combined and tested with some variations, in particular without beads/filters used to grind/enrich pollen. The best approach found in terms of quantity and quality of extracted DNA was a combination of the pretreatment and the extraction method, described in the German guideline, with some modifications and the addition of a DNA purification kit. This protocol was validated with DNA extracts from honey and bee pollen and it was applied to 18 commercial honey samples. Furthermore, a sample proved positive in transgenic screening elements analysis and for transgenic event identification, a knowledge-based approach was adopted. Since the DNA extraction protocol proved suitable, it could be applied for other analysis such as molecular species characterization, the study of traceability, and environmental monitoring, considering honey as a vector of authorized and not authorized genetically modified organisms. [ABSTRACT FROM AUTHOR]

Background: The aim of this study was to identify the Trichomonas vaginalis strains/haplotypes based on identifying their probable variations in asymptomatic patients referred to Tabriz health centers, northwestern Iran. Methods: Sampling was taken from 50-suspected women to T. vaginalis in northwestern Iran. The obtained samples were smeared and cultured. Fifty DNA samples were extracted, amplified and identified by nested polymerase chain reaction and PCR-RFLP of actin gene using two endonuclease enzymes: MseI and RsaI. To reconfirm, the amplicons of actin gene were directly sequenced in order to identify the strains/haplotypes. Results: PCR-RFLP patterns, sequencing and phylogenetic analyses revealed definitely the presence of the G (n=22; 73.4%) and E (n=8; 26.6%) strains. Multiple alignments findings of genotype G showed five haplotypes and two amino acid substitutions in codons 192 and 211 although, no remarkable unique haplotype was found in genotype E. Conclusion: The accurate identification of T. vaginalis strains based on discrimination of their unknown haplotypes particularly those which are impacted on protein translation should be considered in parasite status, drug resistance, mixed infection with HIV and monitoring of asymptomatic trichomoniasis in the region. [ABSTRACT FROM AUTHOR]

[Display omitted] • A specific real-time PCR protocol was developed for faster detection of H. costale. • In France, H. costale was detected in adult oysters since 2005 and spat since 2008. • The H. costale prevalence in spat (14.59%) appeared higher than in adults (6.50%). • Actin gene sequencing showed 2 H. costale strains based on 3 sequence mutations. • The strain 1 was found in hatchery spat while the strain 2 was found in wild spat. The parasite Haplosporidium costale is known to infect and cause mortality in the oyster Crassostrea virginica in the USA. Decades after its first description in the 1960s, this parasite was detected in Crassostrea gigas in the USA and China. However, it presented a low prevalence and no mortality was associated with it. More recently, in 2019, H. costale was detected in France in a batch of moribund oysters. In order to observe how long this parasite has been present on French coasts, from Normandy to Thau lagoon, a retrospective investigation was conducted on 871 adult and spat oyster batches from 2004 to 2020. To allow rapid detection on a large panel of samples, a real-time PCR for the H. costale actin gene was developed. This method allowed the detection of H. costale DNA in adults from 2005 and in spat from 2008. The H. costale prevalence in spat appeared higher than in adults over the years studied, 14.59 % compared to 6.50 %, respectively. All samples presenting positive results were then sequenced on two targets, H. costale rRNA and actin genes. The actin gene sequencing highlighted the presence of two H. costale strains. Adult C. gigas as well as spat batches coming from hatcheries and DNA controls from C. virginica all presented with the Profile 1 H. costale strain. The Profile 2 H. costale strain was detected only in C. gigas spat coming from natural sources. These observations suggest a correlation between the origin of oysters and H. costale strains which may have been caused by commercial imports between Japan, USA and France back to the 1970s. Over the positive samples studied, only few batches (n = 3) suffered mortalities which could be hypothesized to be caused by H. costale , all presenting the Profile 1 H. costale strain. [ABSTRACT FROM AUTHOR]

Background: Trichomonas vaginalis is the agent of urogenital tract infection that causes human trichomoniasis with some serious health complications. More understanding about genetic features of the parasite can be helpful in the study of the pathogenesis, drug susceptibility and epidemiology of the infection. For this end,we conducted analysis of the actin gene of T. vaginalis by applying the PCR-SSCP(PCR-Single Stranded Conformational Polymorphism) and nucleotide sequencingmethod. Methods: Fifty T. vaginalis samples were collected from 950 women attending gynecology clinics in two cities of Iran, Hamadan and Tehran, from November 2010 to July 2011. After axenisation of isolates, all samples subjected to PCR-SSCP and nucleotide sequencing. Results: According to the SSCP banding patterns and nucleotide sequencing, seven sequence types were detected among the isolates. Alignment of the nucleotide sequences showed five polymorphic sites in the different strain types. Amino acid substitution was not observed in the nucleotide sequence translation of the all sequences. Conclusion: The actin gene analysis represents genetic diversity of T. vaginalis and it suggests that various strains can be responsible for clinically different trichomoniasis in infected individuals. It is expected that further studies will be conductedto increase our knowledge about relationship between the actin gene polymorphism and different biological behavior of the parasite.

Faecal samples (363) from kids, lambs, calves and buffalo calves of below 3 months of age were collected from various parts of India and screened microscopically for Cryptosporidium oocysts using modified Ziehl–Neelsen method of staining. Microscopically positive samples (20) were genotyped by PCR amplification of the partial 18S rRNA region and subsequent digestion by SspI, VspI and MboII restriction enzymes. Based on the PCR-RFLP patterns of 18S rRNA, all the 20 samples were found positive for C. parvum. All the positive samples were also used for amplification of partial actin gene of Cryptosporidium spp. For further confirmation of the species of Cryptosporidium, amplified 818 bp partial actin gene of 3 representative isolates was cloned and sequenced. The sequence and phylogenetic analysis of PCR-positive samples confirmed the presence of C. parvum. Thus, actin gene can also be used for specific molecular diagnosis of Cryptosporidium spp., in addition to 18S rRNA. These findings also indicated that young domestic ruminants can be a potent source of cryptosporidial infection for humans and animals in India.

Cryptosporidium is an enteric parasite that infects humans and a wide range of animals. Relatively little is known about the epidemiology and taxonomy of Cryptosporidium in fish. In the present study, a total of 775 fish, belonging to 46 species and comprising ornamental fish, marine fish and freshwater fish were screened for the prevalence of Cryptosporidium by PCR. The overall prevalence of Cryptosporidium in fish was 5.3% (41/775), with prevalences ranging from 1.5 to 100% within individual host species. Phylogenetic analysis of these Cryptosporidium isolates as well as 14 isolates from previous studies indicated extensive genetic diversity as well as evidence for mixed infections. At the 18S locus the following species were identified; Cryptosporidium molnari- like genotype ( n = 14), Cryptosporidium huwi ( n = 8), piscine genotype 2 ( n = 4), piscine genotype 3-like ( n = 1), piscine genotype 4 ( n = 2), piscine genotype 5 ( n = 13), piscine genotype 5-like ( n = 1) and five novel genotypes ( n = 5). At the actin locus, species identification agreed with the 18S locus for only 52.3% of isolates sequenced, indicating high levels of mixed infections. Future studies will need to employ both morphological characterization and deep sequencing amplicon-based technologies to better understand the epidemiological and phylogenetic relationships of piscine-derived Cryptosporidium species and genotypes, particularly when mixed infections are detected. [ABSTRACT FROM AUTHOR]

Trichomonas vaginalis is a human urogenital pathogen that causes trichomoniasis, the most common nonviral, parasitic sexually transmitted infection in the world. At present, little is known regarding the degree of strain variability of T. vaginalis . A classification method for T. vaginalis strains would be a useful tool in the study of the epidemiology, drug resistance, pathogenesis and transmission of T. vaginalis . Eight different types of actin genes have been identified by PCR-RFLP in T. vaginalis ; the purpose of this study is to determine the genotypes of this parasite in Karaj city, Iran. Forty-five clinical T. vaginalis isolates from vaginal secretions and urine sediment were collected from Karaj city from 2012 through 2014. DNA was extracted and the actin gene was amplified by nested-PCR; all samples were positive. To determine the genetic differences, sequencing on seven samples was conducted. Then, all PCR products were digested with HindII, MseI, and RsaI restriction enzymes. Of 45 isolates, 23 samples (51.1%) were of actin genotype G, 11 samples (24.4%) of genotype E, six samples (13.3%) of genotype H, three samples (6.6%) of genotype I, and two samples (4.4%) were mixed genotypes of G and E. Genetic diversity of T. vaginalis isolates is notable. The actin genotype G may be the dominant genotype in Karaj city, Iran. [ABSTRACT FROM AUTHOR]

The morphological, biological, and molecular characteristics of Cryptosporidium piscine genotype 1 from the guppy (Poecilia reticulata) are described, and the species name Cryptosporidium huwi n. sp. is proposed to reflect its genetic and biological differences from gastric and intestinal Cryptosporidium species. Oocysts of C.   huwi n. sp. over-lap in size with Cryptosporidium molnari , measuring approximately 4.4–4.9 µm (mean 4.6) by 4.0–4.8 µm (mean 4.4 µm) with a length to width ratio of 1.04 (0.92–1.35) (n = 50). Similar to C.   molnari , C.   huwi n. sp. was identified in the stomach only and clusters of oogonial and sporogonial stages were identified deep within the epithelium. However, phylogenetic analysis of 18S rRNA sequences indicated that C. huwi n. sp. exhibited 8.5–9.2% and 3.5% genetic distance from C.   molnari isolates and piscine genotype 7 respectively . At the actin locus, the genetic distance between C.   huwi n. sp. and C.   molnari was 16.6%. The genetic distance between C.   huwi n. sp. and other Cryptosporidium species at the 18S locus was 13.2%–17% and at the actin locus was 18.9%–26.3%. Therefore C. huwi n. sp. is genetically distinct from previously described Cryptosporidium species. [ABSTRACT FROM AUTHOR]

Protozoan parasites of the genus Entamoeba infect many classes of vertebrates and are primarily classified based on morphological criteria. To date, only a few species have been proven to cause disease. Here, we examined the pathology of infected pigs with hemorrhage and detected Entamoeba parasites. Isolates were characterized genetically and ultrastructurally to identify the species. Histopathologically, bleeding and thrombus formation were seen only in the large intestine mucosa, where a large number of trophozoites or some Entamoeba cysts were observed around breakdowns in the lamina propria. No screw-shaped bacteria were detected in the lesions, and no pathogenic bacteria such as Brachyspira spp. were detected in fecal cultures. Interestingly, electron microscopy revealed that the parasites possessed mitochondrial organelles, unlike other Entamoeba spp. The isolates were identified as Entamoeba suis by PCR analysis and sequencing of the small subunit ribosomal RNA (SSU rRNA) gene. In phylogenetic analyses based on the actin gene, the E. suis isolate formed a cluster with Entamoeba histolytica and Entamoeba invadens, as well as with other parasites of the Amoebidae. Whether the pathogenicity of the E. suis isolate is affected by the severity of infection or host health status remains unclear; however, our results suggest that E. suis could cause or exacerbate clinical symptoms such as hemorrhagic colitis or diarrhea. [ABSTRACT FROM AUTHOR]

The article discusses the genotype analysis of Cryptosporidium oocysts from fecal samples of asymptomatic kids, lambs and calves in India. Ziehl-Neelsen staining and polymerase chain reaction amplification of 18S rRNA were used to identify infection by the C. parvum protozoa confirming ruminants as vectors for infecting livestock and humans. Comparisons of phylogenetic results were made with the actin gene sequences of specimens from other countries affected by cryptosporidiosis.

Background: Trichomonas vaginalis is the agent of urogenital tract infection that causes human trichomoniasis with some serious health complications. More understanding about genetic features of the parasite can be helpful in the study of the pathogenesis, drug susceptibility and epidemiology of the infection. For this end, we conducted analysis of the actin gene of T. vaginalis by applying the PCR-SSCP (PCR-Single Stranded Conformational Polymorphism) and nucleotide sequencing method. Methods: Fifty T. vaginalis samples were collected from 950 women attending gynecology clinics in two cities of Iran, Hamadan and Tehran, from November 2010 to July 2011. After axenisation of isolates, all samples subjected to PCR-SSCP and nucleotide sequencing. Results: According to the SSCP banding patterns and nucleotide sequencing, seven sequence types were detected among the isolates. Alignment of the nucleotide sequences showed five polymorphic sites in the different strain types. Amino acid substitution was not observed in the nucleotide sequence translation of the all sequences. Conclusion: The actin gene analysis represents genetic diversity of T. vaginalis and it suggests that various strains can be responsible for clinically different trichomoniasis in infected individuals. It is expected that further studies will be conducted to increase our knowledge about relationship between the actin gene polymorphism and different biological behavior of the parasite. [ABSTRACT FROM AUTHOR]

Highlights: [•] A new set of primers for the detection of Cryptosporidium spp. was designed. [•] Cryptosporidium rat genotypes II and III from Brazil and Australia are divergent. [•] Zoonotic transmission of Cryptosporidium spp. by rats in Brazil should be revisited. [•] Cryptosporidium rat genotypes II and III seem to belong to the same species. [ABSTRACT FROM AUTHOR]

Several Heterodera species can reduce the yield of wheat and barley, among which H. avenae, H. filipjevi and H. latipons are economically the most important. Their identification, based on morphological characteristics, is not straightforward but can be made easier using molecular techniques. In this study, we developed species-specific primers for the detection of H. latipons. The actin gene of eight Heterodera species was partially sequenced and, after purifying and sequencing the PCR products, all sequences were aligned to find unique sites. The alignment showed moderate to very high similarities between the species. However, a small fragment of the actin gene was suitable for the construction of a potentially useful species-specific primer for H. latipons. The optimised PCR was subsequently tested with several populations of 14 Heterodera species and a single population of Punctodera punctata. Heterodera latipons was represented by 16 populations originating from six different countries. The primer set (Hlat-act), designed using AllelelD 7.73, was shown to be very specific. To test its sensitivity further, the PCR was conducted on DNA extracted from five second-stage juveniles (J2) of H. latipons mixed with five or 100 J2 belonging to H. avenae. The PCR was able to detect up to 1:10 dilution of the DNA obtained from five J2. The results showed that a specific and sensitive H. latipons species-specific PCR was constructed. [ABSTRACT FROM AUTHOR]

This study describes the isolation, cloning and phylogenetic studies of a partial sequence of actin gene from the fungus Trichoderma harzianum E-58, which was grown in Vogel's medium at 28°C and pH 5.5, with glucose as a carbon source. For the amplification of b-actin gene, the genomic DNA was subjected to the polymerase chain reaction (PCR) by using sequence specific primers. Through agarose gel electrophoresis the amplified PCR product was purified and ligated into pTZ57R/T vector. The ligation mixture was then transformed into E. coli DH10B and spread on ampicillin containing LB agar plate. Restriction analysis was done to confirm the positive transformants. Further, the gene was sequenced, and on the basis of this partial sequence, a dendrogram was made to assess homology among different fungi. These results will surely help study the regulation of cellulase gene expression in the fungus in future. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to determine the prevalence of Cryptosporidium species and genotypes in birds kept in captivity in Brazil. A total of 966 samples from 18 families of birds was collected and stored in 5% potassium dichromate solution at 4°C until processing. Oocysts were purified in Sheather sugar solution following extraction of genomic DNA. Molecular analyses were performed using nested-PCR for amplification of fragments of the 18S subunit of rRNA gene and of the actin gene. Amplification of Cryptosporidium DNA fragments was obtained in 47 (4.86%) samples. Sequencing of amplified fragments and phylogenetic analyses allowed the identification of Cryptosporidium baileyi in a black vulture (Coragyps atratus), a domestic chicken (Gallus gallus domesticus) and a saffron finch (Sicalis flaveola); Cryptosporidium galli in canaries (Serinus canaria), a cockatiel (Nymphicus hollandicus) and lesser seed-finches (Oryzoborus angolensis); Cryptosporidium meleagridis in a domestic chicken (G. g. domesticus); Cryptosporidium parvum in a cockatiel (N. hollandicus); Cryptosporidium avian genotype I in a canary (S. canaria) and an Indian peafowl (Pavo cristatus); Cryptosporidium avian genotype II in ostriches (Struthio camelus) and Cryptosporidium avian genotype III in a cockatiel (N. hollandicus) and a peach-faced lovebird (Agapornis roseicolis). [Copyright &y& Elsevier]

Human trichomoniasis, caused by the protozoan Trichomonas vaginalis, is a highly prevalent sexually transmitted infection. However, little is known about the degree of strain variability of T. vaginalis. A reliable classification method for T. vaginalis strains would be a useful tool in the study of the epidemiology, pathogenesis and transmission of T. vaginalis. A PCR–restriction fragment length polymorphism typing method was designed and evaluated using T. vaginalis isolates obtained after culture of vaginal specimens collected in the Democratic Republic of Congo and in Zambia. The variation of the actin gene of T. vaginalis was determined for three ATCC reference strains and 151 T. vaginalis isolates. Eight different types were identified, on the basis of the digestion patterns of the amplified actin gene, with each of the restriction enzymes HindII, MseI and RsaI . It was determined that the ATCC reference strains 30001, 30240 and 50141 were of actin genotypes G, H and E, respectively. The actin genotype type E was more common in the Democratic Republic of Congo, whereas type G was the commonest type in Zambia. Translation of the nucleotide sequence showed up to three amino acid substitutions. We developed a reproducible, sensitive and specific typing method for T. vaginalis, and were able to distinguish at least eight T. vaginalis actin genotypes. Further studies are needed to evaluate the method using clinical specimens and to determine the utility of the typing method for the genotypic characterization of T. vaginalis. [ABSTRACT FROM AUTHOR]

Bonamia ostreae is a protozoan parasite that infects the European flat oyster Ostrea edulis, causing systemic infections and resulting in massive mortalities in populations of this valuable bivalve species. In this work, we have characterized B. ostreae actin genes and used their sequences for a phylogenetic analysis. Design of different primer sets was necessary to amplify the central coding region of actin genes of B. ostreae. Characterization of the sequences and their amplification in different samples demonstrated the presence of 2 intragenomic actin genes in B. ostreae, without any intron. The phylogenetic analysis placed B. ostreae in a clade with Minchinia tapetis, Minchinia teredinis and Haplosporidium costale as its closest relatives, and demonstrated that the paralogous actin genes found in Bonamia resulted from a duplication of the original actin gene after the Bonamia origin. [ABSTRACT FROM PUBLISHER]

Abstract: An actin gene (named Mar-actin) from the commercial prawn, Macrobrachium rosenbergii, was isolated, sequenced and gene expression was characterized. The cDNA sequence was 1281 bp in length and contained 1131 bp open reading frame encoding 376 amino acids. The amino acid sequence deduced from the nucleotide sequence showed high identity (70.3% to 98.1%) with other known actins of various organisms, highest with that of the European flounder (98.1%). The 3′ untranslated region (3′ UTR) of the Mar-actin mRNA has a high A+U content (approximately 78%) and contains one AUUUUUA and two repeats of the AUUUA motifs, that may function in regulating mRNA decay. Northern blot analysis revealed that the Mar-actin gene was expressed predominantly in muscle tissues. Transcripts in hepatopancreas were barely detectable. Expression of the Mar-actin gene varied during embryonic development and reached the maximal level at the zoea stage. This is the first report describing the complete sequence and expression pattern of the actin gene in prawns. [Copyright &y& Elsevier]

Highly similar gene sequences of the 5′ region of the large subunit (LSU) are commonly interpreted to predict the organism''s identity. However, it was recognised that closely related taxa do not always show sufficiently diverged D1/D2 LSU sequences to differentiate between them. The effectiveness of species separation using D1/D2 LSU sequences, small subunit (SSU) sequences and actin gene sequences was determined by pair-wise comparisons. The LSU data showed coinciding similarities among and within species. The actin data resolved all investigated species. Examples strengthened the value of almost complete SSU sequences for species separation. The larger number of differences in the highly conserved actin gene, compared to the overall more variable LSU gene, is due to the tolerance of protein coding genes to synonymous nucleotide changes. In contrast, the pairing in secondary structures of the rRNA, ensuring the functionality of the molecule, relies on longer and uninterrupted sequence sections. In conclusion, D1/D2 LSU sequences are not specific enough to identify closely related taxa. The actin gene is a better marker in these cases. However, because of the availability of a large database of fungal D1/D2 LSU sequences, this gene region is currently still the preferred target for sequence-based identification. [Copyright &y& Elsevier]

The cell division mechanisms of seven strains from six species of Nannochloris Naumann were analyzed and compared with those of three species of Chlorella Beijerinck and Trebouxia erici Ahmadjian using differential interference microscopy and fluorescence microscopy. Nannochloris bacillaris Naumann divides by binary fission and N. coccoides Naumann divides by budding. Distinct triangular spaces or mother cell walls were found in the dividing autosporangia of the other five strains from four species of Nannochloris , three species of Chlorella , and T. erici. In an attempt to infer an evolutionary relationship between nonautosporic and autosporic species of Nannochloris , we constructed a phylogenetic tree of the actin genes using seven strains from six species of Nannochloris , three species of Chlorella , and T. erici. Nannochloris species were polyphyletic in the Trebouxiophyceae group. Two nonautosporic species of N. bacillaris and N. coccoides were monophyletic and positioned distally. Moreover, to determine their phylogenetic position within the Trebouxiophyceae, we constructed phylogenetic tree of 18S rRNA genes adding other species of Trebouxiophyceae. Nannochloris species were polyphyletic in the Trebouxiophyceae and appeared in two different lineages, a Chlorella–Nannochloris group and a Trebouxia–Choricystis group. The nonautosporic species, N. bacillaris and N. coccoides , and three autosporic species of Nannochloris belonged to the Chlorella–Nannochloris group. Nannochloris bacillaris and N. coccoides were also monophyletic and positioned distally in the phylogenetic tree of 18S rRNA genes. These results suggest that autosporulation is the ancestral mode of cell division in Nannochloris and that nonautosporulative mechanisms, such as binary fission and budding, evolved secondarily. [ABSTRACT FROM AUTHOR]

The cell division mechanisms of seven strains from six species of Nannochloris Naumann were analyzed and compared with those of three species of Chlorella Beijerinck and Trebouxia erici Ahmadjian using differential interference microscopy and fluorescence microscopy. Nannochloris bacillaris Naumann divides by binary fission and N. coccoides Naumann divides by budding. Distinct triangular spaces or mother cell walls were found in the dividing autosporangia of the other five strains from four species of Nannochloris , three species of Chlorella , and T. erici. In an attempt to infer an evolutionary relationship between nonautosporic and autosporic species of Nannochloris , we constructed a phylogenetic tree of the actin genes using seven strains from six species of Nannochloris , three species of Chlorella , and T. erici. Nannochloris species were polyphyletic in the Trebouxiophyceae group. Two nonautosporic species of N. bacillaris and N. coccoides were monophyletic and positioned distally. Moreover, to determine their phylogenetic position within the Trebouxiophyceae, we constructed phylogenetic tree of 18S rRNA genes adding other species of Trebouxiophyceae. Nannochloris species were polyphyletic in the Trebouxiophyceae and appeared in two different lineages, a Chlorella–Nannochloris group and a Trebouxia–Choricystis group. The nonautosporic species, N. bacillaris and N. coccoides , and three autosporic species of Nannochloris belonged to the Chlorella–Nannochloris group. Nannochloris bacillaris and N. coccoides were also monophyletic and positioned distally in the phylogenetic tree of 18S rRNA genes. These results suggest that autosporulation is the ancestral mode of cell division in Nannochloris and that nonautosporulative mechanisms, such as binary fission and budding, evolved secondarily. [ABSTRACT FROM AUTHOR]

A novel one-step method for the differentiation of chicken and turkey DNA is described. The technique uses the polymerase chain reaction (PCR) and primers that exploit intron variability in α-cardiac actin to generate single products of a characteristic size for each species. No cross-reactivity with porcine, ovine or bovine DNA templates is apparent and analysis of chicken/turkey admixtures indicates that it is possible to detect 1% turkey in 99% chicken and vice versa. Because the test is based on a nuclear gene target, it forms a valuable complement to other methods based on mitochondrial DNA sequences. [Copyright &y& Elsevier]

A single cell divides to multiply, but not all cells follow the same pattern of division. We studied cell division in seven strains from six species belonging to the genus Nannochloris Naumann and classified their modes of cell division into three types: binary fission (N. bacillaris Naumann), budding (N. coccoides Naumann), and autosporulation resulting in the formation of two to four daughter cells (N. maculata Butcher, N. sp. SAG 251-2, N. atomus Butcher CCAP 251/7 and SAG 14.87, and N. eucaryotum [Wilhelm et al.] Menzel and Wild). To determine the evolutionary relationships among these multiple modes of cell division, we investigated the strains' genome sizes, copy number of actin genes, and phylogeny. The genome sizes were determined by counter-clamped homogeneous electric fields electrophoresis and fluorimetry. The genomes are very small and range from 12.6 Mbp (N. maculata) to 47.4 Mbp (N. atomus SAG 14.87). The genomes of Nannochloris species seem to be among the smallest for free-living eukaryotes. Nannochloris bacillaris (binary fission), N. coccoides (budding), Nannochloris sp. (two-cell type of autosporulation), and N. eucaryotum (multicell type of autosporulation) contain a single actin gene, whereas N. maculata (two-cell type of autosporulation) and two strains of N. atomus (two-cell type of autosporulation) contain two actin genes. This suggests that the actin gene was duplicated in this eukaryote, which has a very small genome. Phylogenetic analyses of partial actin gene sequences suggest that autosporulation is the ancestral mode of cell division. [ABSTRACT FROM AUTHOR]

Regulation of the vertebrate actin multigene family involves the recognition of various regulatory sequences (cis-acting elements) that specify the distinct tissue type and developmental program of expression for each actin paralogue, which implies that the distribution of cis-acting elements may be unique for each paralogue gene. To elucidate the evolution of these unique distribution patterns, we improved a method to scan for cis-acting elements in the 5′ flanking regulatory region of genes and used it to analyze five cis-acting elements (SRE, MyoD binding site, Elk-1 binding site, positive and negative YY1 binding sites) of six actin paralogue genes (β and γ cytoplasmic actins, α and γ smooth muscle actins, and α skeletal and α cardiac actins) among various vertebrates. It was shown that although an element(s) may exist in all paralogue genes of the same species, its numbers, compositions, and distribution patterns or even sequences vary remarkably among paralogues, which contributes to their different tissue- and developmental-specific expression. However, each pair of coexpressed paralogues has some certain similarity in distribution patterns. Furthermore, among various orthologues of actin genes derived from diverse vertebrates, the sequences, numbers, and distribution patterns of these cis-acting elements are highly conserved or even identical in the long run of phylogeny of vertebrates. Taken together, the results described above strongly indicate that not only the structures of actins but also their expression patterns are essential in both the phylogeny and the physiology of vertebrates. The distribution patterns of cis-acting elements of various actin genes can be regarded as indicators of both horizontal (paralogous) and vertical (orthologous) evolution of actins. [ABSTRACT FROM AUTHOR]

This paper reports the cloning and sequencing of a Phytophthora megasperma actin gene. Sequence analysis revealed that the gene does not contain introns and encodes a putative actin protein remarkably diverged from any other known actins. Genomic Southern analysis indicates that there is a single actin gene per haploid genome, which is actively transcribed in vivo, as revealed by S1 mapping of the transcripts. The transcripts are heterogeneous in size because they have different 3′ termini, none of which contains the conventional AATAAA polyadenylation signal. [ABSTRACT FROM AUTHOR]