[Transgenesis in Worms: Candidates for an Ideal Model].

Transgenesis is an important and often irreplaceable method to study numerous processes of animal life. To create animal transgenic lines, it is necessary to have a suitable model organism that has necessary traits for efficient and affordable transgenesis. The concise review characterizes the existing model organisms of different taxa for which an efficient transgenesis protocol has been developed. Special attention is paid to flatworms and, in particular, Macrostomum lignano as a promising model organism for studying aging, regeneration, and carcinogenesis.

Macrostomum lignano as a model to study the genetics and genomics of parasitic flatworms.

Hundreds of millions of people worldwide are infected by various species of parasitic flatworms. Without treatment, acute and chronical infections frequently lead to the development of severe pathologies and even death. Emerging data on a decreasing efficiency of some important anthelmintic compounds and the emergence of resistance to them force the search for alternative drugs. Parasitic flatworms have complex life cycles, are laborious and expensive in culturing, and have a range of anatomic and physiological adaptations that complicate the application of standard molecular-biological methods. On the other hand, free-living flatworm species, evolutionarily close to parasitic flatworms, do not have the abovementioned difficulties, which makes them potential alternative models to search for and study homologous genes. In this review, we describe the use of the basal free-living flatworm Macrostomum lignano as such a model. M. lignano has a number of convenient biological and experimental properties, such as fast reproduction, easy and non-expensive laboratory culturing, optical body transparency, obligatory sexual reproduction, annotated genome and transcriptome assemblies, and the availability of modern molecular methods, including transgenesis, gene knockdown by RNA interference, and in situ hybridization. All this makes M. lignano amenable to the most modern approaches of forward and reverse genetics, such as transposon insertional mutagenesis and methods of targeted genome editing by the CRISPR/Cas9 system. Due to the availability of an increasing number of genome and transcriptome assemblies of different parasitic flatworm species, new knowledge generated by studying M. lignano can be easily translated to parasitic flatworms with the help of modern bioinformatic methods of comparative genomics and transcriptomics. In support of this, we provide the results of our bioinformatics search and analysis of genes homologous between M. lignano and parasitic flatworms, which predicts a list of promising gene targets for subsequent research.

[A retroposition assay for the NLR1Cth from midge Chironomus thummi genome in the Chinese hamster ovary cells].

Non-Long Terminal Repeats (non-LTR or LINE) retrotransposons belong to the class of mobile genetic elements that are transposed into the host genome by reverse transcription of the RNA intermediate. Most of non-LTR retrotransposons contain two open reading frames (ORFs). The ORF1 codes for a gag-like protein, while the ORF2 codes for a reverse transcriptase (RT). We cloned two constructs based on Jockey-like non-LTR retrotransposon from genome Chironomus thummi (NLR1Cth). The retroposition assay performed in Chinese hamster ovary (CHO) cells demonstrated genome integrations of both constructs. The finding that the insect mobile element NLR1Cth is functional in mammalian cells demonstrates that this element possess universal enzymatic machinery allowing for active propagation in the genome of distant taxa. This suggests that the NLR1Cth transposon system may represent a useful tool for genetic analysis and manipulation in vertebrate cells.

[Somatic mutations of the P53 gene in stomach cancer].

The paper deals with a study of p53 gene somatic mutations in tumor cell genomes from patients with stomach cancers of different histological patterns. It used sequential and molecular cloning methods. The former involved amplicones characterized by abnormal volatility following SSCP analysis of plasmids from 9 tumors. Replacement nucleotides were identified in 4 tumors (intestinal--2, diffuse--2). Among 8 mutations were 1 single-nucleotide deletion in codon-249 with shifting sensing frame and one targeted mutation. Five of the former were missens-mutations which caused amino acid replacement while the other two silent mutations did not. Exon-assisted analysis of p53 ("wild") gene identified cells with stable structure in each tumor (1 mutation--2; 3 mutations--2 including genuinely-paired mutations in 1 exon). All mutations occurred in structurally and functionally important codons. Our evidence corroborated earlier data of SSCP analysis on tumor cell presence in populations with variable p53 genomes.

[The role of the functional sites of the Merlin tumor suppressor in Drosophila spermatogenesis].

The Merlin gene of Drosophila is homologous to the human Neurofibromatosis 2 (NF2) gene an important regulator of proliferation and endocytosis of cell receptors. It was earlier shown that the Thr5 residue of the Drosophila Merlin protein was homologous to Ser518 of the human protein (which was already known to undergo phosphorylation); hence, it was assumed that Thr559 of Drosophila also was a substrate of phosphorylation. The mutant Merlin proteins MerT559D (an analog of the phosphorylated form) and MerT559A (a nonphosphorylated form) were constructed and tested, under the conditions of ectopic expression for the ability to correct the spermatogenesis defects induced by the Mer4 mutation. The mutant form MerT559D was demonstrated to restore the abnormal nebenkern phenotype induced by this mutation, whereas the MerT559A substituted form did not restore this phenotype. Ectopic expression o the wild-type Merlin protein, MerT559A mutant form, and mycMer345-635 truncated protein in a normal genotype resulted in the abnormal nebenkern phenotype, whereas this phenotype was not observed in the case ofectopic expression of the MerT559D analog of the phosphorylated form. Ectopic expression of the mycMer3, mycMerABB, and mycMer-379 truncate variants led to disturbance of meiotic cytokinesis.

[Origin, evolution, and distribution of different groups of non-LTR retrotransposons among eukaryotes].

Non-LTR retrotransposons are an ancient group of retroelements. Twenty-one clades are distinguished today among non-LTR retrotransposons. The presence of different clades in the genome characterizes the diversity of non-LTR retrotransposons of the organism. This review presents a general picture of the evolution and distribution of different clades of non-LTR retrotransposons among the main taxa of eukaryotic organisms: protozoa, plants, fungi, and metazoa. Introduction in the analysis of new taxa and the use of new bioinformatic and experimental approaches can significantly extend our knowledge about non-LTR retrotransposons and their role in the evolution and functioning of eukaryotic genomes.

[Phylogeny of the A genomes of wild and cultivated wheat species].

Diploid species of the genus Triticum L. are its most ancient representatives and have the A genome, which was more recently inherited by all polyploid species. Studies of the phylogenetic relationships among diploid and polyploid wheat species help to identify the donors of elementary genomes and to examine the species specificity of genomes. In this study, molecular analysis of the variable sequences of three nuclear genes (Acc-1, Pgk-1, and Vrn-1) was performed for wild and cultivated wheat species, including both diploids and polyploids. Based on the sequence variations found in the genes, clear differences were observed among elementary genomes, but almost no polymorphism was detected within each genome in polyploids. At the same time, the regions of the three genes proved to be rather heterogeneous in the diploid species Triticum boeoticum Boiss., T. urartu Thum. ex Gandil., and T. monococcum L., thus representing mixed populations. A genome variant identical to the A genome of polyploid species was observed only in T. urartu. Species-specific molecular markers discriminating the diploid species were not found. Analysis of the inheritance of morphological characters also failed to identify a species-specific character for the three diploid wheat species apart from the hairy leaf blade type, described previously.

[LTR retrotransposons from genomes of Aspergillus fumigatus and A. nidulans].

Fungi Aspergillus spp. are able to infect all tissues and organs and often cause invasive mycosis (aspergillosis), which is usually a fatal disease, especially in the patients with compromised immune system. Microbiological monitoring of these infectious agents is necessary in modem medical facilities. Mobile elements can be used as markers for identification of species and strains of Aspergillus found indoors as well as in aspergillosis diagnostics. Genomic sequences of two representative Aspergillus species, A. fumigatus and A. nidulans, were analysed in silico in order to detect LTR retrotransposons. We found considerable differences in the composition of retrotransposon families between two studied species. One of the detected families, which is present in both studied Aspergillus species, is phylogenetically quite different from all other known fungal retrotransposons. The majority of elements are represented by damaged copies. Nevertheless, we describe for the first time allegedly non-damaged LTR copies that contain intact ORFs and could be active.

[Homology-dependent inactivation of LTR retrotransposons in genomes of Aspergillus fumigatus and A. nidulans].

Repeat-induced point mutation (RIP) is the most intriguing among the known mechanisms of repeated sequences inactivation because of its ability to produce irreversible mutation of repeated DNA. Discovered for the first time in Neurospora crassa, RIP is characterized by C:G to T:A transitions in duplicated sequences. The mechanisms and distribution of RIP are still purely investigated. Mobile elements are a common target for the processes which lead to homology-dependent silencing because of their ability to propagate themselves. We have done comparative analysis of LTR retrotransposons in genomic scale from genomes of two aspergilli fungi--Aspergillus funmigatus and A. nidulans, based on several copies we reconstructed "de-RIP" retroelements. Investigations of frequencies of CpG, CpA and TpG sites, which are potential targets for mutagenesis, showed the much lower frequencies of these sites in mobile elements in comparison with structural genes. LTR retrotransposons from A. fumigatus and A. nidulans have different ratio of types of substitutions. Our analysis indicates that two investigated fungi have or had the RIP-like processes for repeated sequences inactivation, in various modes. Whereas in A. fumigatus the context for mutagenesis consists of both CpG and CpA sites, in A. nidulans inactivation seems to proceed only on CpG dinucleotides. The present investigation gives a theoretical background for planning of experimental studying of RIP inactivation in aspergilli.

[Structure and expression of the F6.2 gene in Chironomus thummi and other Chironomus species]. / Struktura i ékspressiia gena F6.2 u Chironomus thummi i drugikh vidov roda Chironomus.

A full-length copy of the F6.2 gene from the tissue-specific BRa locus of the Chironomus thummi chromosome IV was isolated and analyzed. The gene contains two exons (715 and 644 bp, respectively) and one 172-bp intron. The data of the RT-PCR analysis demonstrated that F6.2 was transcriptionally active at different developmental stages of Chironomus thummi and at least in the last larval stage of C. dorsalis. The distribution of the F6.2 gene among 42 species of Chironomus, as well as among two other genera of the family Chironomidae was examined by means of PCR. The F6.2 sequence was found in 34 Chironomus species. Using in situ hybridization, three species were analyzed for the presence of the F6.2 homologous sequences. In five species, the sequence of the F6.2 PCR product was determined. In these species, the intron size polymorphism caused by the variation of the number of the intron-forming repeats was observed. The data obtained provided evaluation of the F6.2 distribution among the genus Chironomus.

[Molecular differences in Adh1-F and Adh1-S alleles in the sugar beet Beta vulgaris L]. / Molekuliarnye razlichiia allelei Adh1-F i Adh1-S u sakharnoi svekly Beta vulgaris L.

We compared nucleotide sequences of exon 4 and part of exon 5 of alleles F and S of the Adh1 locus controlling alcohol dehydrogenase in sugar beet. The Adh1-F and Adh1-S sequences of the examined fragment were shown to differ by two nucleotides. Adenine (A) and cytosine (C) of Adh1-F were substituted by respectively thymine (T) and adenine (A) in Adh1-S. Consequently, glutamine and asparagine from the F subunit of ADH1 are replaced by valine and lysine, respectively. Because of differences in the amino acid content, the F subunit is by two elementary charges more negatively charged electrically than the S subunit, which correlates with differences in their electrophoretic mobility. Comparison of the examined Adh1 fragment of sugar beet with its counterparts in other plants showed that the sites bearing substitutions in the former species are classed as variable.

[Phylogenetic relationships among holarctic populations of Chironomus entis and Chironomus plumosus in view of possible horizontal transfer of mitochondrial genes]. / Filogeneticheskie vzaimootnosheniia golarkticheskikh populiatsii Chironomus entis i Chironomus plumosus s uchetom vozmozhnoi gorizontal'noi peredachi mitokhondrial'nykh genov.

In eight Holarctic populations of two typical chironomid sibling species of the plumosus group, Chrionomus entis and Chironomus plumosus, nucleotides sequences of mitochondrial (cytb) and nuclear (gb2b) gene regions were examined. The phylogenetic trees reflecting the evolutionary histories of the nuclear and mitochondrial markers exhibited significant differences. On the tree based on the nuclear gene sequences the populations clustered according to their species affiliation, whereas on the tree based on the mitochondrial gene sequences the populations were grouped according to their geographic position. This discrepancy is probably explained by mitochondrial gene flow between sympatric species with incomplete reproductive isolation (sibling species). Based on our results together with the earlier data on nuclear and mitochondrial gene sequences of some other species from the phylogenetic group plumosus, a scheme of phylogenetic relationships within this group is proposed. This scheme is in many ways different from the traditional view on the evolutionary relationships among species of the plumosus group.

[Headcase gene--proliferation or hormonal control?]. / Gen headcase--proliferatsiia ili gormonal'nyi kontrol'?

A new insertion allele of the hdc gene was isolated and described. The nucleotide sequence of the coding region had no detectable homology with genomic DNA of any other Drosophila species, except for D. mauritiana. Gene expression was found both in adult testes and ovaries and at embryonic and larval stages. This expression pattern exhibits a strong similarity to that of cell-cycle genes. In contrast to cycE (a typical cell-cycle gene), which leads to expression termination after in vivo culturing of the wing disk, hdc did not arrest expression. It is concluded that hdc is a species-specific differentiation gene, whose regulatory activity in the development of an organism differs from that of proliferation genes.

Structure and polymorphism of the Chironomus thummi gene encoding special lobe-specific silk protein, ssp160.

cDNA encoding Chironomus thummi ssp160 was used to isolate a genomic clone that hybridized in situ to band A2b on polytene chromosome IV, the site of the ssp160 gene. DNA sequencing, primer extension and gene/cDNA nucleotide sequence alignment revealed the gene contains six exons and five introns; 70% of ssp160 is encoded in exon 3. Variations between cDNA and gene sequences led to the design of a polymerase chain reaction, restriction fragment length polymorphism assay that was subsequently used to demonstrate the existence of polymorphic alleles whose distribution varied between geographically separated populations of larvae. The polymorphism is associated with codon deletions in a six-amino-acid repeat containing an N-linked glycosylation motif. These deletions may have resulted from slipped-strand mispairing during DNA replication.

The Chironomus (Camptochironomus) tentans genome contains two non-LTR retrotransposons.

A cDNA library from salivary gland cells of Chironomus tentans was screened with a probe containing the NLRCth1 non-LTR (long terminal repeat) retrotransposon from Chironomus thummi. Several positive clones were obtained and one of them, p62, was characterized by in situ hybridization and sequencing. The sequencing analysis showed that this clone contained a 4607 bp nucleotide sequence of a new transposable element that hybridized in situ to more than 100 sites over all four C. tentans chromosomes. The detailed analysis of this sequence revealed the presence of the 3'-end of open reading frame 1 (ORF1), a complete ORF2, and a 1.3-kb 3'-end untranslated region (UTR). The new element has been designated NLRCt2 (non-LTR retrotransposon 2 from C. tentans). A comparison of the nucleotide sequences of NLRCth1 and NLRCt2 showed 30% similarity in the region of ORF1 and 70% similarity in the region of ORF2. Based on the results of Southern blot analysis, two transposable elements have been found in the C. tentans genome, one of which is identical to NLRCth1 from C. thummi. This may be explained by horizontal transmission. The second element, NLRCt2, has been found in two different forms in the C. tentans genome. These can be distinguished by the presence of the 1.3-kb 3'-end UTR in one of the forms. Since the cDNA clone investigated was isolated from a tissue-specific cDNA library, the data showed that NRLCt2 is expressed in somatic cells.

The Chironomus thummi genome contains a non-LTR retrotransposon.

Nineteen recombinant phages containing DNA from the region of Balbiani ring a (BRa), which develops on chromosome IV in cells of the special lobe of the Chironomus thummi salivary gland, were isolated from a Chironomus thummi genomic library. Three of the clones contained transposable element sequences that hybridized to more than 100 sites on all four Chironomus chromosomes, including constant and variable sites. Two handogous clones, lambda 24 (which lacks the transposable element) and lambda 43 (which contains this insertion) were investigated by nucleotide sequence analysis. The complete nucleotide sequence of the 4.8 kb transposable element from Chironomus thummi (NLR1Cth) is reported here. This element contains two overlapping open reading frames of 1887 (ORF1) and 2649 bp (ORF2). Three cysteine motifs are found in the sequence of ORF1. Sequence similarity was found between ORF2 and known genes of viruses and transposable elements which encode reverse transcriptase. The NLR1Cth element has no long terminal repeats and is flanked by short direct repeats of the sequence TATCACTGACAAC. A 24 bp poly(dA) sequence was found at the 3' end of the element. Based upon its structural organization and comparative analysis of its nucleotide sequence we suggest that this NLR1Cth element belongs to the class of non-LTR retrotransposons. The genomic clone pC6.10 was previously obtained by microdissection and cloning of DNA from polytene chromosome IV of Chironomus thummi. A 2.4 kb insertion contained part of the 3' terminal region of the NLR1Cth element, but this differed in sequence from the first copy by several nucleotide substitutions and a shorter poly (dA) tract at the 3' end.(ABSTRACT TRUNCATED AT 250 WORDS)

MEC: a transposable element from Chironomus thummi (diptera).

Two genomic clones, pC1.2 and p20D (containing inserts of 2.0 and 1.6 kb, respectively) were isolated from the A2b region to polytene chromosome IV of Chironomus thummi thummi salivary gland cells. Upon in situ hybridization to polytene chromosomes of C. thummi thummi and C. thummi piger, p20D DNA hybridized mainly over the A2b region of chromosome IV, whereas pC1.2 DNA hybridized to at least 90 sites distributed over all the chromosomes. A partial nucleotide sequence analysis showed that these clones were very similar and allowed the detection of a 596 bp insert in the pC1.2 clone. This insert possesses all of the essential features of a Class II transposable element and was called MEC. It carries a nearly perfect 107 bp terminal inverted repeat containing one mismatch and is flanked by a 5 bp direct repeat. The 372 bp central region contains a short open reading frame with a coding capacity of 58 amino acids.