Molecular Characterization of Acanthamoeba spp. Occurring in Water Bodies and Patients in Poland and Redefinition of Polish T16 Genotype

Acanthamoeba genus is divided into 20 genotypes (T1–T20) on the basis of the gene encoding 18S rRNA sequence. Using of at least 2 kbp gene fragments is strongly recommended to identify new genotypes and 5% difference is commonly used as a criterion of new genotypes, however, this value is questionable. In this paper, Polish Acanthamoeba strains described earlier on the basis of ~850 bp Ami fragment of 18S rRNA gene as T4, T11 and a new T16 genotype, have been analyzed using near‐complete sequence of the gene. This analysis was needed because the Ami fragment does not reveal full variability within 18S rRNA gene. Phylogenetic analysis based on Ami fragment is biased by artifacts in the construction of the tree, so the fragment should not be used for identification of new putative Acanthamoeba genotypes. The analysis confirmed that the Polish sequences represent T4 and T11 genotypes and that the strains described earlier as T16 genotype are in fact a new subgroup of the T20 genotype and that this genotype should be divided into two subgroups: T20a (two strains described by [J. Eukaryot. Microbiol. 62 (2015) 69]) and T20b (11 Polish strains described in this study). The T20b subgroup was isolated from both clinical samples and water bodies used by people as bathing places and there is a risk of infection for humans during contact with water.     

A compact unc45b‐promoter drives muscle‐specific expression in zebrafish and mouse

Summary: Gene therapeutic approaches to cure genetic diseases require tools to express the rescuing gene exclusively within the affected tissues. Viruses are often chosen as gene transfer vehicles but they have limited capacity for genetic information to be carried and transduced. In addition, to avoid off‐target effects the therapeutic gene should be driven by a tissue‐specific promoter in order to ensure expression in the target organs, tissues, or cell populations. The larger the promoter, the less space will be left for the respective gene. Thus, there is a need for small but tissue‐specific promoters. Here, we describe a compact unc45b promoter fragment of 195 bp that retains the ability to drive gene expression exclusively in skeletal and cardiac muscle in zebrafish and mouse. Remarkably, the described unc45b promoter fragment not only drives muscle‐specific expression but presents heat‐shock inducibility, allowing a temporal and spatial quantity control of (trans)gene expression. Here, we demonstrate that the transgenic expression of the smyd1b gene driven by the unc45b promoter fragment is able to rescue the embryonically lethal heart and skeletal muscle defects in smyd1b‐deficient flatline mutant zebrafish. Our findings demonstrate that the described muscle‐specific unc45b promoter fragment might be a valuable tool for the development of genetic therapies in patients suffering from myopathies. genesis 54:431–438, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.     

Structure and expression of three src2 homologues and a novel subfamily of flavoprotein monooxygenase genes revealed by the analysis of a 25 kb fragment from Arabidopsis thaliana chromosome IV

Biological and computer-assisted analyses of a 25 kb fragment from Arabidopsis thaliana chromosome IV led to the characterization of two multigene families and three novel orphan genes, not previously described. The first gene family named AtMO1-4 encodes monooxygenases, related to the prokaryotic salicylate hydroxylases. The second gene family contains three members, two on the analysed 25 kb fragment and one on chromosome I. The latter three genes lack introns and are homologous to the previously studied Glycine max src2 gene which is overexpressed at low temperature. Gene expression and primary structure of the deduced proteins are described and compared. Three genes of unknown function, showing tissue specific expressions, are characterized on the 25 kb fragment. Full length or partial cognate cDNAs have been sequenced for all the genes studied.     

Lambdoid phages that simplify the recovery of in vitro recombinants

Summary Derivatives of phage λ are described for use as vectors for fragments of DNA generated with theHindIII andEcoRI restriction enzymes. With some vectors, hybrid molecules are recognised by a change from a turbid to a clear plaque morphology resulting from the insertion of a fragment of DNA into the λ gene coding for the phage regressor. Other vectors contain a central, replaceable fragment of DNA which imparts a readily recognisable phenotype. This central fragment may include either a gene for a mutant transfer RNA (suppressor) or a part of thelacZ gene ofE. coli able to complement alacZ host. The appropriatelacZ host and indicator plates permit the ready distinction between recombinant and vector phages by the colour of the plaques.     

In vitro constructed plasmids containing both ends of bacteriophage Mu DNA express phage functions

Summary The construction of a plasmid carrying the right end PstI·B fragment of bacteriophage Mu DNA and of plasmids containing in addition the left end EcoRI·C fragment of Mu DNA into the vector pBR322 is described. Inversion of the G segment still occurs in all these plasmids. By marker rescue and complementation experiments the right PstI cleavage site was located to the left of gene Q. The composite plasmids inheriting also the left end EcoRI fragment of Mu DNA express both the immunity and killing functions of Mu and direct the in vitro synthesis of presumably Mu-specific polypeptides. These results demonstrate that Mu-specific functions can be analyzed from cloned fragments.     

New evidence of the genus Homo from East Rudolf, Kenya. I

Three new fossil hominid specimens that were recovered in 1970 from the Plio-Pleistocene sediments to the east of Lake Rudolf are described. They include the left side of the body and the symphyseal region of an adult mandible that contains three molar teeth (KNM-ER 730), an edentulous left-sided mandibular fragment (KNM-ER 731) and the shaft of a left femur (KNM-ER 737). The specimens are described in anatomical detail, illustrated and selected measurements given. It is concluded that they should be attributed to the genus Homo sp. indet. Detailed comparative studies will be published in due course.     

The subfossil monkey femur and subfossil monkey tibia of the Antilles: A review

The proximal portion of a subfossil monkey femur found in a Jamaican cave shares all the femoral characters of a mature male Cebus apella.The fragment alone, however, does not prove conspecificity. The Jamaican femur is also of a size that could belong to the extinct Xenothrix mcgregoriof the same island. In contrast, the distal portion of a monkey tibia recovered from a kitchen midden in the Dominican Republic cannot be identified with that of any known living platyrrhine or catarrhine monkey. Geological age, geographic locality, and size of fragment point to probable alignment of the tibia with the recently extinct cebid Saimiri bernensis.Although no conclusive identifications are made, the distinctive characters of the two limb bones are described on the basis of comparisons with femurs and tibias representing all genera of living platyrrhines, most genera of catarrhine monkeys, and some strepsirhines.     

Nematode‐specific PCR primers for the 18S small subunit rRNA gene

A set of polymerase chain reaction primers were designed, which amplify a c. 1 kb fragment of the 18S ribosomal DNA gene, and are specific to the phylum Nematoda. These have proven useful in isolating nematode genes from samples mixed with other biological material, particularly with application to DNA barcoding. Optimal reaction conditions are described. These primers have successfully amplified the correct fragment from a wide phylogenetic range of nematodes, and have so far generated no sequences from any other organismal group.     

First record ofColoborhynchus (Pterosauria) from the Santana Formation (Lower Cretaceous) of the Chapada do Araripe, Brazil

The anterior tips of associated upper and lower jaws of a pterosaur from the Lower Cretaceous of Brazil are described and assigned to the taxonColoborhynchus in the family Ornithocheiridae. It is characterized by the shape and position of the sagittal crest on the upper and lower jaw, the arrangement and length of the teeth and the spoon-like lateral expansion of the anterior parts of the jaws. It closely resemblesColoborhynchus wadleighi from North America andColoborhynchus clavirostris from England. Diagnostic anatomical characteristics permit a revision of the genusTropeognathus, which is shown here to be a junior synonym of other described taxa.Tropeognathus mesembrinus is referred toCriorhynchus andT. robustus toColoboryhnchus. Consistent anatomical features enable the new jaw fragment to be assigned toColoborhynchus robustus.      

Identification of three RFLPs at the HCK locus on chromosome 20

New HindIII, RsaI and TaqI restriction fragment length polymorphisms (RFLPs) within the haemopoietic cell kinase gene in chromosome band 20q11.2 are described. These RFLPs provide a useful marker for linkage analysis in proximal 20q.     

DNA Sequence of Genes for Detoxification of Fusaric Acid, a Wilt-inducing Agent Produced by Fusarium Species

Isolation and nucleotide sequence determination of fusaric acid-detoxification genes are described in this paper. For screening the genes, bacteria collected from soil were positively selected in a selective medium containing fusaric acid. The capability of fusaric acid-resistant isolates to detoxify the toxin was assayed by examining the survival of tomato callus cells in culture filtrates prepared from the bacterial culture, in the presence of fusaric acid. The isolate (HY-1) showing the highest detoxification was selected and identified as Klebsiella oxytoca. Chromosomal DNA of this isolate was digested with Bam HI and shotgun-cloned to fusaric acid-sensitive E. coli. The DNA fragment carrying fusaric acid-detoxification genes was further shortened by enzyme digestion and the open reading frames in the fragment were analyzed by determining total nucleotide sequences of the fragment. Finally, three open reading frames were shown to be essential for expressing the detoxification of fusaric acid. These frames possessed a single promoter sequence at the upstream region of the first open reading frame. Northern blot analysis showed that these genes were polycistronically transcribed to express the fusaric acid detoxification, strongly supporting th results of DNA sequence analysis.     

Differentiation of Chlamydia Species by Combined Use of Polymerase Chain Reaction and Restriction Endonuclease Analysis

To differentiate Chlamydia spp., a primer pair designed to generate a genus-specific region of the major outer membrane protein (MOMP) gene was used in a PCR to amplify a single DNA fragment of 245-259 bp. In the PCR, the expected single DNA fragment was amplified from strains of Chlamydia trachomatis, C. psittaci, C. pneumoniae and C. pecorum, respectively. By restriction endonuclease analysis with AluI and PvuII, the amplified products exhibited four distinct patterns, corresponding to the four species. It is, therefore, concluded that one-step PCR followed by restriction endonuclease analysis as described in this study could be a valuable method for the detection and differentiation of Chlamydia species.     

An efficient plasmid vector for expression cloning of large numbers of PCR fragments in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The described plasmid pEamTA was designed for parallel polymerase chain reaction (PCR) cloning of open reading frames (ORFs) in Escherichia coli. It relies on the well-known TA-cloning principle, and the “T-vector” can be generated from a plasmid preparation by digestion with the restriction enzyme Eam1105I. The single 3′-T-overhangs of the vector fragment are positioned in a way that A-tailed PCR-products beginning with the start-ATG of an ORF end up in optimal position for expression from a strong tac-promoter when ligated in correct orientation. The orientation of the insert can be checked via a reconstituted NdeI site (catATG) present in correct clones. The protocol works regardless of internal restriction sites of the PCR fragment, a major advantage when cloning a number of fragments in parallel. It also does not require 5′-primer extensions and finally delivers an expression clone for the preparation of untagged protein in less than a week.     

New evidence of the genus Homo from East Rudolf,Kenya (III)

A new fossil hominid partial skeleton (KNM-ER 803) that was discovered from the Plio-Pleistocene sediments to the east of Lake Rudolf is described. It includes parts of a femur, two tibiae, an ulna, two radii, a third metatarsal and several toe bones. There are also two teeth, an upper canine and an upper central incisor. A second new fossil hominid (KNM-ER 164) is represented by a parietal fragment, two vertebrae and some hand bones. A third is represented by a massive left femur (KNM-ER 999). The specimens are described in anatomical detail, some are illustrated and selected measurements are given. It is concluded that they should be attributed to the genus Homo sp. indet. Detailed comparative studies will be published in due course.     

A rapid,single leaf,nucleic acid assay for determining the cytoplasmic organelle complement of rapeseed and related Brassica species

Summary An assay is described whereby Eco RI restriction fragment length polymorphisms of mitochondrial and chloroplast DNAs can definitively identify cytoplasms of interest in Brassica crop development. Restrictable mitochondrial and chloroplast DNA is extracted from as little as 2–3 g and 0.5 g leaf tissue, respectively, and the donor plants are able to continue to develop in a normal manner. An unknown cytoplasm can be identified in three days, which is a considerable saving in time and labor compared to the several years required by traditional methods. The assay is very inexpensive and should be established as a routine procedure in laboratories involved in sexual or somatic Brassica hybrid production.     

Distribution of restriction site polymorphism within the chloroplast genome of the genus Glycine,subgenus Soja

Summary Restriction fragment length polymorphisms (RFLPs) have been used to detect intragenic sequence diversity in Glycine subgenus soja chloroplast DNA. The distribution of these RFLPs allow Glycine max and G. soja accessions to be grouped according to cytoplasmic genetic relatedness. DNA clones from mung bean chloroplast DNA were used to locate the RFLPs to specific regions of the chloroplast genome. In the course of the experiments, several previously unobserved RFLPs were also identified. At least six molecular changes were detected, including both restriction site loss or gain and insertion/deletion events. Three of the fragment polymorphisms detected are due to changes in the juncture region between one inverted repeat region and the large single-copy region. Probes detecting polymorphisms in three representative soybean genotypes were used to screen additional cultivars and Plant Introductions. The distribution of RFLP patterns in these accessions were consistent with the patterns of previously described cytoplasmic groupings, with the exception of one accession, which formed a new plastome group.     

Isolation and characterization of a gene encoding meso-diaminopimelate dehydrogenase fromGlycine max

A detailed characterization of the lysine biosynthetic pathway in plants is yet to be completed. It is, however, assumed that the diaminopimelic acid pathway exists in the plant kingdom, as commonly described forEscherichia coli. Modification and refinement of lytic complementation, a technique previously utilized in bacterial systems, facilitated the isolation of a functional Diaminopimelate Dehydrogenase gene from aGlycine max nuclear gene library. The isolated gene codes for the enzyme meso-diaminopimelate dehydrogenase. The coding capacity for the enzyme was originally contained on a 6.6kb fragment in a Charon 4a soybean gene bank. Subcloning of the 6.6kb fragment resulted in the recombinant plasmid pMW75. Subsequent subcloning resulted in a 4.05 kb fragment contained in pLW14. One region of homology was observed upon hybridization to EcoR1 digested soybean DNA. Homologous sequences were also observed in Triticum DNA. Meso-diaminopimelate dehydrogenase activity was demonstrated inGlycine max embryos. Maximum enzymatic activity of the cloned enzyme was observed at a pH of 8.0. The enzyme encoded by the soybean gene has an apparent molecular weight of 67 000.     

Mini-chromosomes: plasmids which carry the E. coli replication origin.

Summary We have isolated plasmids by linking the 5.9 MD EcoRI fragment of E. coli that carries the origin of replication to an EcoRI fragment that carries an ampicillin resistance determinant, but lacks an origin of replication. 3 plasmids of this type, pOC1, pOC2, and pOC3, are described in detail in this report. Although the plasmids have some adverse effect on the growth properties of the host strain, their existence shows that two functioning chromosomal origins can coexist in one cell.Deletions generated from this type of plasmids allow an allocation of the origin of replication of E. coli within a DNA segment less than 0.4 MD in size.