Transgene-free genome editing in marine algae by bacterial conjugation - comparison with biolistic CRISPR/Cas9 transformation.

The CRISPR/Cas9 technology has opened the possibility for targeted genome editing in various organisms including diatom model organisms. One standard method for delivery of vectors to diatom cells is by biolistic particle bombardment. Recently delivery by conjugation was added to the tool-box. An important difference between these methods is that biolistic transformation results in transgene integration of vector DNA into the algae genome, whereas conjugative transformation allows the vector to be maintained as an episome in the recipient cells. In this study, we have used both transformation methods to deliver the CRISPR/Cas9 system to the marine diatom Phaeodactylum tricornutum aiming to induce mutations in a common target gene. This allowed us to compare the two CRISPR/Cas9 delivery systems with regard to mutation efficiency, and to assess potential problems connected to constitutive expression of Cas9. We found that the percentage of CRISPR-induced targeted biallelic mutations are similar for both methods, but an extended growth period might be needed to induce biallelic mutations when the CRISPR/Cas9 system is episomal. Independent of the CRISPR/Cas9 vector system, constitutive expression of Cas9 can cause re-editing of mutant lines with small indels. Complications associated with the biolistic transformation system like the permanent and random integration of foreign DNA into the host genome and unstable mutant lines caused by constitutive expression of Cas9 can be avoided using the episomal CRISPR/Cas9 system. The episomal vector can be eliminated from the diatom cells by removal of selection pressure, resulting in transient Cas9 expression and non-transgenic mutant lines. Depending on legislation, such lines might be considered as non-GMOs.

Characterization and evolution of a myrosinase from the cabbage aphid Brevicoryne brassicae.

The aphid myrosinase gene has been elucidated using Rapid Amplification of cDNA Ends-PCR. Sequencing has shown that aphid myrosinase has significant sequence similarity (35%) to plant myrosinases and other members of glycosyl hydrolase family 1 (GHF1). The residues acting as proton donor and nucleophile, in the hydrolysis of glucosinolates by aphid myrosinase, are identified as Glu 167 and Glu 374 respectively. The equivalent residues in plant myrosinase are Gln 187 and Glu 409 and for the cyanogenic beta-glucosidase Glu 183 and Glu 397. Thus it would appear that the absence of a proton donor is not necessary for the hydrolysis of glucosinolates as was thought to be the case for the plant myrosinases. Aphid myrosinase appears to be more similar to animal beta-O-glucosidases than to plant myrosinases, as assessed by sequence similarity and phylogenetic techniques. These results strongly suggest that myrosinase activity has twice arisen from beta-O-glucosidases in plants and animals. Comparison of aphid myrosinase with plant myrosinase has highlighted Lys 173 and Arg 312 as possibly playing a crucial role in the hydrolysis of glucosinolates by aphid myrosinase.

Expression of homologues for p53 and p73 in the softshell clam (Mya arenaria), a naturally-occurring model for human cancer.

Homologues for human p53 (Hsp53) and p73 (Hsp73) genes were cloned and expression patterns for their corresponding proteins analysed in tissues from normal and leukemic softshell clams (Mya arenaria). These are the first structural and functional data for p53 and p73 cDNAs and gene products in a naturally occurring, non-mammalian disease model. Core sequence of the predicted clam p53 (Map53) and p73 (Map73) proteins is virtually identical and includes the following highly conserved regions: the transcriptional activation domain (TAD), MDM2 binding site, ATM phosphorylation site, proline rich domain, DNA binding domains (DBDs) II-V, nuclear import and export signals and the tetramerization domain. The core sequence is a structural mosaic of the corresponding human proteins, with the TAD and DBDs resembling Hsp53 and Hsp73, respectively. This suggests that Map53 and Map73 proteins may function similarly to human proteins. Clam proteins have either a short (Map53) or long (Map73) C-terminal extension. These features suggest that Map53 and Map73 may be alternate splice variants of a p63/p73-like ancestral gene. Map73 is significantly upregulated in hemocytes and adductor muscle from leukemic clams. In leukemic hemocytes, both proteins are absent from the nucleus and sequestered in the cytoplasm. This observation suggests that a non-mutational p53/p73-dependent mechanism may be involved in the clam disease. Further studies of these gene products in clams may reveal p53/p73-related molecular mechanisms that are held in common with Burkitt's lymphoma or other human cancers.

Genetic structure and evolution of RAC-GTPases in Arabidopsis thaliana.

Rho GTPases regulate a number of important cellular functions in eukaryotes, such as organization of the cytoskeleton, stress-induced signal transduction, cell death, cell growth, and differentiation. We have conducted an extensive screening, characterization, and analysis of genes belonging to the Ras superfamily of GTPases in land plants (embryophyta) and found that the Rho family is composed mainly of proteins with homology to RAC-like proteins in terrestrial plants. Here we present the genomic and cDNA sequences of the RAC gene family from the plant Arabidopsis thaliana. On the basis of amino acid alignments and genomic structure comparison of the corresponding genes, the 11 encoded AtRAC proteins can be divided into two distinct groups of which one group apparently has evolved only in vascular plants. Our phylogenetic analysis suggests that the plant RAC genes underwent a rapid evolution and diversification prior to the emergence of the embryophyta, creating a group that is distinct from rac/cdc42 genes in other eukaryotes. In embryophyta, RAC genes have later undergone an expansion through numerous large gene duplications. Five of these RAC duplications in Arabidopsis thaliana are reported here. We also present an hypothesis suggesting that the characteristic RAC proteins in higher plants have evolved to compensate the loss of RAS proteins.

Prevalence of GB virus C (also called hepatitis G virus) markers in Norwegian blood donors.

GB virus C (GBV-C), also called hepatitis G virus (HGV), occurs worldwide, but the clinical significance of this virus is still unclear. Plasma samples from 1,001 blood donors were tested by reverse transcription PCR using primers from the NS5 region and by a commercial enzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin G antibodies against the putative envelope of HGV (anti-HGV E2). GBV-C/HGV RNA was present in the plasma from 2.5% of the blood donors, and anti-HGV E2 antibodies could be detected in 10.5% of the samples. Only one of the blood donors with viremia had elevated levels of alanine aminotransferase. Among ELISA-positive donors, there was a significantly higher percentage (16.5%) of individuals who had been treated by acupuncture than individuals who had not been given this treatment (9.4%). No other variables showed significant differences. Screening of medical records from 401 recipients of blood from PCR-positive donors revealed no association with liver disease. Four of 12 partners (33%) were HGV RNA positive, and sequence analyses of the strains showed that four of the couples probably were infected with the same strains, while strains from different couples were not identical. Anti-HGV E2 antibodies were detected in serum samples from four other partners. The prevalence of GBV-C/HGV among blood donors in our region is dramatically higher than the prevalence of hepatitis C virus (0.03%).

The Drosophila Ral GTPase regulates developmental cell shape changes through the Jun NH(2)-terminal kinase pathway.

The Ral GTPase is activated by RalGDS, which is one of the effector proteins for Ras. Previous studies have suggested that Ral might function to regulate the cytoskeleton; however, its in vivo function is unknown. We have identified a Drosophila homologue of Ral that is widely expressed during embryogenesis and imaginal disc development. Two mutant Drosophila Ral (DRal) proteins, DRal(G20V) and DRal(S25N), were generated and analyzed for nucleotide binding and GTPase activity. The biochemical analyses demonstrated that DRal(G20V) and DRal(S25N) act as constitutively active and dominant negative mutants, respectively. Overexpression of the wild-type DRal did not cause any visible phenotype, whereas DRal(G20V) and DRal(S25N) mutants caused defects in the development of various tissues including the cuticular surface, which is covered by parallel arrays of polarized structures such as hairs and sensory bristles. The dominant negative DRal protein caused defects in the development of hairs and bristles. These phenotypes were genetically suppressed by loss of function mutations of hemipterous and basket, encoding Drosophila Jun NH(2)-terminal kinase kinase (JNKK) and Jun NH(2)-terminal kinase (JNK), respectively. Expression of the constitutively active DRal protein caused defects in the process of dorsal closure during embryogenesis and inhibited the phosphorylation of JNK in cultured S2 cells. These results indicate that DRal regulates developmental cell shape changes through the JNK pathway.

Identification of a C-->T mutation in the reactive-site coding region of the C1-inhibitor gene and its detection by an improved mutation-specific polymerase chain reaction method.

Mutations in the C1-inhibitor (C1-INH) gene, leading to low functional levels of C1-inhibitor protein, cause hereditary angioedema (HAE). The disease is characterized by episodic edema in a number of organs. Typically, swellings occur in extremities and face, often accompanied by crampy abdominal pain. Laryngeal edema may lead to suffocation. Type II HAE patients have low functional C1-INH values stemming from only one normal allele. Antigenic C1-INH values, however, are normal or increased owing to the presence of a dysfunctional protein from the mutated allele. The mutations are usually found in exon 8 coding for the amino acids near the reactive centre (P1). Previously, no mutations in the C1-INH gene had been published from the Scandinavian countries. In this work, exon 8 of the C1-inhibitor gene was sequenced in members of two different kindreds, from western and northern Norway, who were suffering from HAE type II. A common point mutation was found within the bait region encoding the reactive centre. The codon CGC was converted to TGC at position 17970, corresponding to an Arg-->Cys replacement which reportedly is the second most frequent type II HAE mutation. This information was utilized to develop a mutation-specific polymerase chain reaction (PCR) for the identification of affected family members. The antisense 17-mer primer (5'-AAGACCAGCAGGGTGCA-3') was successfully applied and AmpliTaq Gold was used in the PCR.

Cloning and characterization of rac-like cDNAs from Arabidopsis thaliana.

The Rho family of GTPases are in higher eukaryotes divided into 3 major subfamilies; the Rho, Rac and Cdc42 proteins. In plants, however, the Rho family is restricted to one large family of Rac-like proteins. From work with mammalian phagocytes the Rac proteins are known to activate a multicomponent NADPH-dependent oxidase which results in accumulation of H2O2, a process termed oxidative burst. In plants a similar oxidative burst is observed and plays and important role in its defence against pathogen infections, suggesting a similar role for the plant Rac-like proteins. The Rho family of GTPases proteins are also involved in control of cell morphology, and are also thought to mediate signals from cell membrane receptors. In a broad search for members of the Ras superfamily in plants, several new small GTP-binding proteins were found. We report here the identification and molecular cloning of 5 rac-like cDNAs from Arabidopsis thaliana, Arac1-5. The Rac-like proteins deduced from the cDNA sequences all share 80-95% homology, but show considerably more diversity on the nucleotide level, indicating that this is an ancient gene family. Four of the rac genes were found to be expressed in all tissues examined, but one gene, Arac2, was expressed exclusively in the root, hypocotyl and stem. Our results show that the rac gene family in A. thaliana consists of at least 10 different genes.

Detection of cytomegalovirus using 'boosted' nested PCR.

As a part of a study of an outbreak of CMV infections in a neonatal care intensive care unit, a modified nested PCR was developed for detection of CMV DNA in clinical specimens. Standard nested PCR involves a critical step; passage of PCR products from the first reaction round to the second round. We have adapted a 'boosted' nested PCR which implies amplification in one single step, thus reducing the contamination problems. Nasopharyngeal aspirates and urine samples from patients with perinatal CMV infections, breast milk from some of their mothers, amniotic fluids, urine samples and lymphocytes from seropositive healthy adults were examined by PCR and culture. In the total of 614 of clinical specimens, the PCR test yielded positive results in 51 samples from 14 patients, whereas CMV was isolated in 25 samples from 11 cases only. All samples from healthy individuals were negative. CMV DNA was detected in all culture-positive samples, but all samples from healthy adults were negative. 29/68 culture negative specimens were positive by PCR. No cross-reactivity to other herpes viruses or to human DNA was observed. Our findings show a high sensitivity and a high specificity of the 'boosted' nested PCR. We conclude that the described PCR method can be used for the rapid detection of CMV in clinical specimens with a greatly reduced risk of contamination, and it has proved to be a very useful tool in diagnostic work.

Mutational analysis of the carboxy-terminal portion of p53 using both yeast and mammalian cell assays in vivo.

Increasing evidence indicates that p53 is a transcriptional trans-activator through its sequence-specific DNA binding domain. Tumor-derived p53 mutations disrupt the trans-activation ability mainly due to loss of its sequence-specific DNA binding. Using both yeast and mammalian cell assays, the effect of p53 mutations in the carboxy terminal portion was investigated in order to address how p53 mutations outside of the DNA binding domain affect p53 function. The p53 cDNA in the carboxy-terminus was randomly mutagenized by error-prone polymerase chain reactions and the amplified cDNA was screened for the ability to trans-activate using a yeast assay. Four p53 mutations, including two missense and two nonsense mutations located in the carboxy-terminal oligomerization domain, were further analysed for trans-activation, cell cycle arrest and colony formation in a human osteosarcoma cell line, Saos-2. These functional properties of p53 were disrupted by the missense mutations. Surprisingly, one of the nonsense mutations disrupts the trans-activation function and the ability to G1 arrest but shows a strong inhibition of colony formation. These results confirm that mutations in the oligomerization domain can inactivate p53 function and also indicate that p53-mediated cell growth inhibition does not necessarily depend on the ability to arrest cell cycle.

The myrosinase (thioglucoside glucohydrolase) gene family in Brassicaceae.

The glucosinolate hydrolyzing enzymes myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) are encoded by a multigene family consisting of two subgroups. The first two nuclear genes representing each of these two subgroups of the new gene family, Myr1.Bn1 and Myr2.Bn1, from Brassica napus have been cloned and sequenced. Based on conserved regions in cDNA of three species, PCR (polymerase chain reaction) primers were made, and used to amplify and characterize the structure of the myrosinase genes in seven species of Brassicaceae. Southern hybridization analysis of PCR products and genomic DNA indicates that myrosinase is encoded by at least 14 genes in B. napus, with similar numbers in the other species of Brassicaceae investigated. The Myr1 gene cloned from B. napus has a 19 amino acid signal peptide and consists of 11 exons of sizes ranging from 54 to 256 bp and 10 introns of sizes from 75 to 229 bp. The Myr2 gene has a 20 amino acid signal peptide and consists of 12 exons ranging in size from 35 to 262 bp and 11 introns of sizes from 81 to 131 bp. The exons from the two genes have 83% homology at the amino acid level. The intron-exon splice sites are of GT..AG consensus type. The signal peptides and presence of sites for N-linked glycosylation, suggest transport and glycosylation through the ER-Golgi complex. The differences between the two genes are discussed on the basis of their predicted expression at different developmental stages in the plant. Both genes show homology to a conserved motif representing the glycosyl hydrolase family of enzymes.

Isotope inequilibrium of glucose metabolites in intact cells and particlefree supernatants of Ehrlich ascites tumor.

With an enzyme degradative technique, isotope inequilibrium of glucose metabolites was demonstrated in intact cells and particlefree supernatants of Ehrlich ascites tumor using 1-14C-glucose as tracer. Inequilibrium was found between glucose and glucose-6-phosphate, glucose and fructose-6-phosphate, glucose and 6-phosphogluconate, while glucose-6-phosphate were found to be in near-equilibrium within the incubation time investigated. Glucose and lactate were found to be in near equilibrium after 8 min in intact cells. Calculations based on the equilibrium levels found, showed that these inequilibria could not be explained by the effects of the pentose cycle.