SS Genome Research Articles

Evolution of the S-Genomes in Triticum-Aegilops Alliance: Evidences From Chromosome Analysis.

Five diploid Aegilops species of the Sitopsis section: Ae. speltoides, Ae. longissima, Ae. sharonensis, Ae. searsii, and Ae. bicornis, two tetraploid species Ae. peregrina (= Ae. variabilis) and Ae. kotschyi (Aegilops section) and hexaploid Ae. vavilovii (Vertebrata section) carry the S-genomes. The B- and G-genomes of polyploid wheat are also the derivatives of the S-genome. Evolution of the S-genome species was studied using Giemsa C-banding and fluorescence in situ hybridization (FISH) with DNA probes representing 5S (pTa794) and 18S-5.8S-26S (pTa71) rDNAs as well as nine tandem repeats: pSc119.2, pAesp_SAT86, Spelt-1, Spelt-52, pAs1, pTa-535, and pTa-s53. To correlate the C-banding and FISH patterns we used the microsatellites (CTT)10 and (GTT)9, which are major components of the C-banding positive heterochromatin in wheat. According to the results obtained, diploid species split into two groups corresponding to Emarginata and Truncata sub-sections, which differ in the C-banding patterns, distribution of rDNA and other repeats. The B- and G-genomes of polyploid wheat are most closely related to the S-genome of Ae. speltoides. The genomes of allopolyploid wheat have been evolved as a result of different species-specific chromosome translocations, sequence amplification, elimination and re-patterning of repetitive DNA sequences. These events occurred independently in different wheat species and in Ae. speltoides. The 5S rDNA locus of chromosome 1S was probably lost in ancient Ae. speltoides prior to formation of Timopheevii wheat, but after the emergence of ancient emmer. Evolution of Emarginata species was associated with an increase of C-banding and (CTT)10-positive heterochromatin, amplification of Spelt-52, re-pattering of the pAesp_SAT86, and a gradual decrease in the amount of the D-genome-specific repeats pAs1, pTa-535, and pTa-s53. The emergence of Ae. peregrina and Ae. kotschyi did not lead to significant changes of the S*-genomes. However, partial elimination of 45S rDNA repeats from 5S* and 6S* chromosomes and alterations of C-banding and FISH-patterns have been detected. Similarity of the Sv-genome of Ae. vavilovii with the Ss genome of diploid Ae. searsii confirmed the origin of this hexaploid. A model of the S-genome evolution is suggested.

Single stranded adeno-associated virus achieves efficient gene transfer to anterior segment in the mouse eye.

Adeno-associated viruses (AAVs) are used extensively as a gene delivery vehicle for retinal gene therapy, yet its ability to target the anterior segment of the eye, critical to unlocking therapeutic opportunities, is less characterized. Previously, self-complimentary (sc) AAV was shown to be necessary for transduction of the cornea and trabecular meshwork (TM), limiting the size of the gene transfer cassette, likely due to a block in second strand synthesis thought to be required for functional transduction. Here, we evaluated several AAV capsids in a single stranded (ss) genome conformation for their ability to overcome the need for scAAV for targeting corneal endothelium and TM. AAV2, 8, and a recently synthetically developed AAV called Anc80L65 were evaluated in vitro and in vivo by intracameral injection in mice. Results show that although scAAV2 demonstrated superior infectivity in vitro including Human Trabecular meshwork (HTM) immortalized cell lines; Anc80L65 transduced following a single intracameral injection efficiently all components of the mouse anterior segment, including the TM, corneal stroma, and endothelial cells. These results suggest that Anc80L65 is able to overcome the requirement for scAAV genomes to enable TM and corneal targeting, expanding the potential experimental and therapeutic use of AAV gene transfer in the anterior segment of the eye.

Specific growth rate and multiplicity of infection affect high-cell-density fermentation with bacteriophage M13 for ssDNA production.

The bacteriophage M13 has found frequent applications in nanobiotechnology due to its chemically and genetically tunable protein surface and its ability to self-assemble into colloidal membranes. Additionally, its single-stranded (ss) genome is commonly used as scaffold for DNA origami. Despite the manifold uses of M13, upstream production methods for phage and scaffold ssDNA are underexamined with respect to future industrial usage. Here, the high-cell-density phage production with Escherichia coli as host organism was studied in respect of medium composition, infection time, multiplicity of infection, and specific growth rate. The specific growth rate and the multiplicity of infection were identified as the crucial state variables that influence phage amplification rate on one hand and the concentration of produced ssDNA on the other hand. Using a growth rate of 0.15 h-1 and a multiplicity of infection of 0.05 pfu cfu-1 in the fed-batch production process, the concentration of pure isolated M13 ssDNA usable for scaffolded DNA origami could be enhanced by 54% to 590 mg L-1 . Thus, our results help enabling M13 production for industrial uses in nanobiotechnology. Biotechnol. Bioeng. 2017;114: 777-784. © 2016 Wiley Periodicals, Inc.

Genomic analyses reveal recurrent mutations in epigenetic modifiers and the JAK-STAT pathway in Sézary syndrome.

Sézary syndrome (SS) is an aggressive leukaemia of mature T cells with poor prognosis and limited options for targeted therapies. The comprehensive genetic alterations underlying the pathogenesis of SS are unknown. Here we integrate whole-genome sequencing (n=6), whole-exome sequencing (n=66) and array comparative genomic hybridization-based copy-number analysis (n=80) of primary SS samples. We identify previously unknown recurrent loss-of-function aberrations targeting members of the chromatin remodelling/histone modification and trithorax families, including ARID1A in which functional loss from nonsense and frameshift mutations and/or targeted deletions is observed in 40.3% of SS genomes. We also identify recurrent gain-of-function mutations targeting PLCG1 (9%) and JAK1, JAK3, STAT3 and STAT5B (JAK/STAT total ∼11%). Functional studies reveal sensitivity of JAK1-mutated primary SS cells to JAK inhibitor treatment. These results highlight the complex genomic landscape of SS and a role for inhibition of JAK/STAT pathways for the treatment of SS.

Integrated Genome Sequencing Reveals Frequent Loss of Function Alterations of ARID1A and Other Epigenetic Modifiers in Sezary Syndrome

IntroductionSezary Syndrome (SS) is an aggressive mature T-cell neoplasm characterized by erythroderma, generalized lymphadenopathy and circulating T-cells. SS cells are characterized by complex karyotypes with numerous structural alterations and copy-number variants. Previous studies have implicated disruption and/or haploinsufficiency of TP53, CDKN2A, RB1 and PTEN in SS. Nevertheless, the comprehensive genetic alterations underlying the pathogenesis of SS are unknown. In this study, we used an integrated genomic sequencing approach to characterize the genetic basis of SS.MethodsSS cases that fulfilled established diagnostic criteria including characteristic cytologic, immunophenotypic and karyotypic features were analyzed. We performed genomic analysis on 84 primary SS cases using whole genome sequencing (WGS; n = 6), high-resolution array comparative genome hybridization (aCGH using NimbleGen 270K Feature Array; n = 80) and whole exome sequencing (WES) using Illumina TruSeq Exome Capture (n = 66). We filtered this combined dataset to identify 463 genes within regions of recurrent copy-number variations that were also impacted by protein coding mutations. We focused on 16 of these genes and confirmed 75 mutations in these genes by Sanger sequencing. For 35 of these mutations, normal tissue was available to confirm somatic acquisition.ResultsWGS revealed a complex structural genome with numerous regions of chromothrypsis and aneuploidy with over 1,000 inter- and intra-chromosomal translocations in the 6 SS genomes but did not reveal recurrent structural alterations. Array CGH identified many aneuploidies reported previously including 8+, 10-, 17p-/iso17 and 19q-. These results were corroborated by aCGH analysis which also revealed recurrent genomic losses at 9p21.3 (CDKN2A), 10p11.22 (PTEN) and 13q14.2 (RB1). Importantly, exome sequencing further identified several novel somatic, deleterious mutations in these three genes. Strikingly, in addition to confirming loss of function alterations of genes previously implicated in SS pathogenesis, we identified genetic alterations in genes involved in epigenetic modification including chromatin/histone modification (including ARID1A, ARID5B, MLL, MLL2, MLL4, SETD1A, SETD1B, SETD6) and DNA methylation (including NCOR1, TET1, TET2, DNMT3A, DNMT3B) not previously implicated in SS pathogenesis.Among the most frequent and most restricted regions of recurrent deletion was the 1p36.11 region. Of the 18 genes within this region, ARID1A showed the highest frequency of disruption (27/80 showing deletion; 7/66 showing novel mutations including a frameshift mutation at residue 1449 within the GR binding domain of ARID1A). Of the SS genomes for which both WES and aCGH analysis were performed, 25/62 (40.3%) showed disruption of ARID1A either by deletion or mutation or both. Moreover ARID5B was also targeted by deletions of region 10q21.2 in 23/80 genomes and 3/66 SS genomes showed mutations. We also identified a narrow region of recurrent aneuploidy affecting the SWI/SNF family-member of SMARC genes known to interact with ARID-family SMARCC1at 3p21.31. This gene was recurrently targeted by a deletion in 17/80 (21.3%) SS genomes by highly restricted deletional events (<4Mb).Altogether, deletions and/or mutations in ARID1A, ARID5B or SMARCC1 were identified in 38 of 62 (61.3%) of SS genomes. Further analysis revealed that other members of the ARID- and SMARC-families, ARID1B, ARID2 and ARID4A, were mutated. Moreover, ARID3A and SMARCA4 (both located on 19p) were targeted by recurrent deletions in 29/80 and 18/80 SS genomes, respectively. Altogether, deletions and/or mutations in at least one of the ARID- or SMARC-family genes were identified in 91.9% of SS.ConclusionThis study is the first to comprehensively profile the genomic landscape in SS. Integration of high-resolution aCGH with mutational analysis reveals alterations in epigenetic regulators through loss-of-function lesions in ARID- and SMARC-family genes in over 90% of SS. The results of our study offer further insights into the pathogenesis of SS and provide support for use of drugs targeting deregulated chromatin remodeling pathways in the treatment of SS patients. DisclosuresNo relevant conflicts of interest to declare.

The chloroplast view of the evolution of polyploid wheat.

Polyploid wheats comprise four species: Triticum turgidum (AABB genomes) and T. aestivum (AABBDD) in the Emmer lineage, and T. timopheevii (AAGG) and T. zhukovskyi (AAGGA(m) A(m) ) in the Timopheevi lineage. Genetic relationships between chloroplast genomes were studied to trace the evolutionary history of the species. Twenty-five chloroplast genomes were sequenced, and 1127 plant accessions were genotyped, representing 13 Triticum and Aegilops species. The A. speltoides (SS genome) diverged before the divergence of T. urartu (AA), A. tauschii (DD) and the Aegilops species of the Sitopsis section. Aegilops speltoides forms a monophyletic clade with the polyploid Emmer and Timopheevi wheats, which originated within the last 0.7 and 0.4 Myr, respectively. The geographic distribution of chloroplast haplotypes of the wild tetraploid wheats and A. speltoides illustrates the possible geographic origin of the Emmer lineage in the southern Levant and the Timopheevi lineage in northern Iraq. Aegilops speltoides is the closest relative of the diploid donor of the chloroplast (cytoplasm), as well as the B and G genomes to Timopheevi and Emmer lineages. Chloroplast haplotypes were often shared by species or subspecies within major lineages and between the lineages, indicating the contribution of introgression to the evolution and domestication of polyploid wheats.

The Characterization of RNA Viruses in Tropical Seawater Using Targeted PCR and Metagenomics

ABSTRACTViruses have a profound influence on the ecology and evolution of plankton, but our understanding of the composition of the aquatic viral communities is still rudimentary. This is especially true of those viruses having RNA genomes. The limited data that have been published suggest that the RNA virioplankton is dominated by viruses with positive-sense, single-stranded (+ss) genomes that have features in common with those of eukaryote-infecting viruses in the order Picornavirales (picornavirads). In this study, we investigated the diversity of the RNA virus assemblages in tropical coastal seawater samples using targeted PCR and metagenomics. Amplification of RNA-dependent RNA polymerase (RdRp) genes from fractions of a buoyant density gradient suggested that the distribution of two major subclades of the marine picornavirads was largely congruent with the distribution of total virus-like RNA, a finding consistent with their proposed dominance. Analyses of the RdRp sequences in the library revealed the presence of many diverse phylotypes, most of which were related only distantly to those of cultivated viruses. Phylogenetic analysis suggests that there were hundreds of unique picornavirad-like phylotypes in one 35-liter sample that differed from one another by at least as much as the differences among currently recognized species. Assembly of the sequences in the metagenome resulted in the reconstruction of six essentially complete viral genomes that had features similar to viruses in the families Bacillarna-, Dicistro-, and Marnaviridae. Comparison of the tropical seawater metagenomes with those from other habitats suggests that +ssRNA viruses are generally the most common types of RNA viruses in aquatic environments, but biases in library preparation remain a possible explanation for this observation.

Comparative genomic in situ hybridization (cGISH) analysis of the genomic relationships among Sinapis arvensis, Brassica rapa and Brassica nigra

To further understand the relationships between the SS genome of Sinapis arvensis and the AA, BB genomes in Brassica, genomic DNA of Sinapis arvensis was hybridized to the metaphase chromosomes of Brassica nigra (BB genome), and the metaphase chromosomes and interphase nucleus of Brassica rapa (AA genome) by comparative genomic in situ hybridization (cGISH). As a result, every chromosome of B. nigra had signals along the whole chromosomal length. However, only half of the condensed heterochromatic areas in the interphase nucleus and the chromosomes showed rich signals in Brassica rapa. Interphase nucleus and the metaphase chromosomes of S. arvensis were simultaneously hybridized with digoxigenin-labeled genomic DNA of B. nigra and biotin-labeled genomic DNA of B. rapa. Signals of genomic DNA of B. nigra hybridized throughout the length of all chromosomes and all the condensed heterochromatic areas in the interphase nucleus, except chromosome 4, of which signals were weak in centromeric regions. Signals of the genomic DNA of B. rapa patterned the most areas of ten chromosomes and ten condensed heterochromatic areas, others had less signals. The results showed that the SS genome had homology with AA and BB genomes, but the homology between SS genome and AA genome was clearly lower than that between the SS genome and BB genome.

Nucleic acid packaging in viruses

We review recent literature describing protein nucleic acid interactions and nucleic acid organization in viruses. The nature of the viral genome determines its overall organization and its interactions with the capsid protein. Genomes composed of single strand (ss) RNA and DNA are highly flexible and, in some cases, adapt to the symmetry of the particle-forming protein to show repeated, sequence independent, nucleoprotein interactions. Genomes composed of double-stranded (ds) DNA do not interact strongly with the container due to their intrinsic stiffness, but form well-organized layers in virions. Assembly of virions with ssDNA and ssRNA genomes usually occurs through a cooperative condensation of the protein and genome, while dsDNA viruses usually pump the genome into a preformed capsid with a strong, virally encoded, molecular motor complex. We present data that suggest the packing density of ss genomes and ds genomes are comparable, but the latter exhibit far higher pressures due to their stiffness.

Inter-sectional hybrids obtained from reciprocal crosses between &lt;i&gt;Begonia semperflorens&lt;/i&gt; (section &lt;i&gt;Begonia&lt;/i&gt;) and &lt;i&gt;B.&lt;/i&gt; ‘Orange Rubra’ (section &lt;i&gt;Gaerdita&lt;/i&gt; × section &lt;i&gt;Pritzelia&lt;/i&gt;)

Inter-sectional hybrids were successfully obtained by the reciprocal crosses between 11 cultivars (including 6 diploids and 5 tetraploids) of Begonia semperflorens (SS & SSSS genomes) and B. ‘Orange Rubra’ (RR genome) with the aid of in vitro culture of mature or immature seeds on MS medium containing 0.1 mg l−1 α-naphthylacetic acid, 0.1 mg l−1 6-benzyladenine, 10 mg l−1 gibberellic acid, 30 g l−1 sucrose and 2.5 g l−1 gellan gum. Embryo rescue as ovary culture with immature seeds 12th–16th day after pollination (DAP) generally gave higher efficiency of plantlet formation, but in some cross combinations, culture of mature seeds (30 DAP) resulted in higher yield of plantlets. Flow cytometric analysis revealed that they were consisted of the plants with various genomic combinations (RS, RR, RSS, RRS, RRSS and RRRRSS) as estimated by the DNA contents of both parents. Hybridity of these plants with various genomic combinations including RR was confirmed by random amplified polymorphic DNA analysis. These results suggested that unreduced gamete formation and spontaneous chromosome doubling were involved in the hybrid formation of various ploidy levels and genomic combinations. These hybrids showed various levels of intermediate traits between both parents according to the genomic compositions, and some of them had desirable characters of both parents.

Cloning and Characterization of Genes Coding for Fructan Biosynthesis Enzymes (FBEs) in Triticeae Plants

Fructan is not only a carbon source for storage but also plays an important role as anti-stress agents in many plant species. Complex fructans having both β-(2,1)- and β-(2,6)-linked fructosyl units accumulate in Triticeae plants commonly. Three enzymes (sucrose: sucrose 1-fructosyltransferase, 1-SST, EC: 2.4.1.99; sucrose: fructan 6-fructosyltransferase, 6-SFT, EC: 2.4.1.10; and fructan: fructan 1-fructosyltransferase, 1-FFT, EC: 2.4.1.100) were involved in fructan biosynthesis in Triticeae plant species. We successfully isolated these genes from tetraploid wheat ( Triticum turgidum, genotype: AABB), common wheat ( Triticum aestivum L., genotype: AABBDD) and three wild relatives of common wheat, Triticum urartu Thum. (the origin of the AA genome), Aegilops speltoides (Tausch) Gren. (the putative source of the SS genome) and Aegilops tauschii Coss. (the source of the DD genome). Sequence analysis revealed that all the FBEs (fructan biosynthetic enzymes) had three highly conserved functional motifs except 1-SST (EU981912) from tetraploid wheat species only with conserved DPNG. Low pI (isoelectric point) and potential N-glycosylation sites were predicted, which were crucial for protein compartmentation and post-translational process. Analysis on subcelluar localization signals showed that only 6-SFT had vacuolar-directed signal. Sequences alignment result showed that 1- SST and 1- FFT were more conservative and had closer relationship each other, while 6- SFT was more active during the evolution processing. According to the syntenic relationship between wheat and rice genome, FBEs were predicated to be located on the homeologous group 6 and group 2 chromosomes. Expression profile confirmed that expression of all the three FBEs were drought-stress induced. This study can assist to establish a useful theoretical platform for cold- or drought-tolerant improvement of wheat by modulating FBEs expression.

Divergent evolution of wild and cultivated subspecies of Triticum timopheevii as revealed by the study of PolA1 gene

Triticum timopheevii (genome symbol AAGG) comprises two subspecies, cultivated ssp. timopheevii, and wild ssp. armeniacum. These two subspecies are considered as allotetraploids of AA genome from Triticum diploid species and SS genome from Aegilops species. The difference in genome symbol (G vs. S) is due to wide genetic variations among four SS genome species, Ae. bicornis, Ae. longissima, Ae. searsii, and Ae. speltoides. In order to study the origin of T. timopheevii, we compared 19th intron (PI19) sequence of the PolA1 gene, encoding the largest subunit of RNA polymerase I. Two different sized DNA fragments containing PI19 sequences (PI19A and PI19G) were amplified both in ssp. timopheevii and ssp. armeniacum. Shorter PI19A (112 bp) sequences of both subspecies were identical to PI19 sequences of two AA species, T. monococcum and T. urartu. Interestingly, the longer PI19G (241–243 bp) sequences of ssp. armeniacum showed more similarity to PI19 sequences of Ae. speltoides whereas ssp. timopheevii showed more similarity to PI19 sequences of other three SS genome species. The results indicated that two subspecies of T. timopheevii, ssp. armeniacum or ssp. timopheevii, might have arisen independently by allotetraploidization of AA genome with Ae. speltoides or one of the remaining three Aegilops species, respectively.

Efficient Intrathymic Gene Transfer Following In Situ Administration of a rAAV Serotype 8 Vector in Mice and Nonhuman Primates

The thymus is the primary site of T-cell development and plays a key role in the induction of self-tolerance. We previously showed that the intrathymic (i.t.) injection of a transgene-expressing lentiviral vector (LV) in mice can result in the correction of a T cell-specific genetic defect. Nevertheless, the efficiency of thymocyte transduction did not exceed 0.1-0.3% and we were unable to detect any thymus transduction in macaques. As such, we initiated studies to assess the capacity of recombinant adeno-associated virus (rAAV) vectors to transduce murine and primate thymic cells. In vivo administration of AAV serotype 2-derived single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors pseudotyped with capsid proteins of serotypes 1, 2, 4, 5, and 8 demonstrated that murine thymus transduction was significantly enhanced by scAAV2/8. Transgene expression was detected in 5% of thymocytes and, notably, transduced cells represented 1% of peripheral T lymphocytes. Moreover, i.t. administration of scAAV2/8 particles in macaques, by endoscopic-mediated guidance, resulted in significant gene transfer. Thus, in healthy animals, where thymic gene transfer does not provide a selective advantage, scAAV2/8 is a unique tool promoting the in situ transduction of thymocytes with the subsequent export of gene-modified lymphocytes to the periphery.

Aegilops section Sitopsis species contains the introgressive PolA1 gene with a closer relationship to that of Hordeum than Triticum-Aegilops species

The section Sitopsis in the genus Aegilops includes five species, Ae. speltoides, Ae. longissima, Ae. sharonensis, Ae. searsii, and Ae. bicornis, which share the SS genome. Although extensive molecular studies have indicated Ae. speltoides as a donor of BB or GG genome to polyploid wheat species, the precise relationships among SS, BB, and GG genomes remain unclear. PolA1 is a single-copy nuclear gene encoding the largest subunit of RNA polymerase I. Highly polymorphic PolA1 exon 20 sequences were analyzed for 11 Triticum–Aegilops, 13 Hordeum and three related species. Phylogenetic analyses of the PolA1 gene showed that Triticum–Aegilops and Hordeum species were distinctly separated into two clades. Two related species, Secale cereale and Dasypyrum villosum, were grouped into Triticum and Hordeum clades, respectively. Interestingly, seven accessions of the Sitopsis species were clustered into the Hordeum clade whereas two accessions belonged to the Triticum clade. In contrast, all accessions of Sitopsis species shared the same haplotype of plastid PSID sequences with Triticum–Aegilops species. This inconsistency in phylogeny between nuclear and cytoplasmic sequences suggested that the Sitopsis species probably originated through introgressive hybridization between ancestral species of Triticum–Aegilops and Hordeum.

Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver

AbstractTransduction with recombinant adeno-associated virus (AAV) vectors is limited by the need to convert its single-stranded (ss) genome to transcriptionally active double-stranded (ds) forms. For AAV-mediated hemophilia B (HB) gene therapy, we have overcome this obstacle by constructing a liver-restricted mini–human factor IX (hFIX) expression cassette that can be packaged as complementary dimers within individual AAV particles. Molecular analysis of murine liver transduced with these self-complementary (sc) vectors demonstrated rapid formation of active ds-linear genomes that persisted stably as concatamers or monomeric circles. This unique property resulted in a 20-fold improvement in hFIX expression in mice over comparable ssAAV vectors. Administration of only 1 × 1010 scAAV particles led to expression of hFIX at supraphysiologic levels (8I U/mL) and correction of the bleeding diathesis in FIX knock-out mice. Of importance, therapeutic levels of hFIX (3%-30% of normal) were achieved in nonhuman primates using a significantly lower dose of scAAV than required with ssAAV. Furthermore, AAV5-pseudotyped scAAV vectors mediated successful transduction in macaques with pre-existing immunity to AAV8. Hence, this novel vector represents an important advance for hemophilia B gene therapy.

101. Molecular Mechanism Responsible for High Level Transgene Expression in Murine and Nonhuman Primate Liver with the Second Generation of Novel AAV Serotype Vectors

AAV vectors are being considered for in vivo application of gene therapy in the treatment of a variety of disorders. This study evaluates the biology of second generation vectors based on the novel serotypes AAV7 and 8 containing self-complimentary (sc) genomes in both murine nonhuman primate livers. Superior murine liver transduction by sc over the traditional single stranded (ss) vector genomes was confirmed for all serotypes examined. Stable levels of transgene expression were achieved in cynomolgus macaques with efficiencies at least 2 logs higher than what was achieved with AAV2 vectors using ss genomes. In an attempt to understand molecular mechanisms responsible for the higher level gene transfer, we performed a series of detailed molecular studies of the vector genomes. This included treatment with exonuclease with specific substrate preferences, single cutter restriction enzyme digestion mediated and locus specific hybridization based vector genome mapping, bacteriophage Phi29 DNA polymerase mediated double stranded circular template specific isothermal rolling circle linear amplification for rescue, cloning and characterization of persisted circular genomes, and differential quantification of different populations of vector genomes by real time PCR. Our data indicated that the higher level of transgene expression achieved with the sc vectors was due to both increased numbers of persisting genomes and increased activity of these genomes. Study of the molecular dynamics of vector genome processing in murine liver revealed that both ss and sc vectors persisted as episomal circular forms but sc vectors were more quickly and more efficiently converted into circular forms. The structure and formation of concatemers were similar with the types of genomes. The molecular structures of AAV genomes in macaques were more heterogeneous consisting of primarily circular monomer and concatemers in different configurations, and some unexpected molecular entities. More extensive rearrangements and deletions were observed in macaque than in mouse. It will be useful to further understand differences in the molecular processing of AAV genomes in mice and macaques as human applications are being considered.

Variation in the distribution of a genome-specific DNA sequence on chromosomes reveals evolutionary relationships in the Triticum and Aegilops complex

The present study analyzed the distribution pattern of the Ae. speltoides–derived repetitive clone pGc1R-1 in the Triticum/Aegilops complex. Fluorescence in situ hybridization analysis showed that clone pGc1R-1 is a S-genome-specific repetitive sequence that hybridized to the S-genome of three species in the section Sitopsis, Aegilops speltoides (S), Ae. longissima (Sl), and Ae. sharonensis (Ssh), but not to Ae. bicornis (Sb) and Ae. searsii (Ss), nor to any other diploid Aegilops species. This clone also hybridized to the very closely related G-genome of T. timopheevii subsp. armeniacum and T. timopheevii ssp. timopheevii, but not to the B-genome of T. turgidum and T. aestivum. Hybridization also was observed in the polyploid Aegilops species, Ae. kotschyi (UkSk), Ae. peregrina (UpSp), and Ae. vavilovii (XvaDvaSva). Large inter- and intraspecific variations were observed. Our results confirm that the S genome is related more to the Sl and Ssh genomes than to the Sb and Ss genomes; there is a greater affinity between the G and S genomes than between the B and S genomes. Mechanisms to account for the variation in the FISH pattern with different genomes include sequence amplification and deletion. Variation in the distribution of this genome-specific DNA sequence, pGc1R-1, on chromosomes can be used to reveal evolutionary relationships in the Triticum and Aegilops complex.

Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo.

Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Following portal-vein administration of rAAV vectors in vivo, single-stranded (ss) rAAV genomes become double stranded (ds), circularized, and/or concatemerized concomitant with a slow rise and, eventually, steady-state levels of transgene expression. Over time, at least some of the stabilized genomes become integrated into mouse chromosomal DNA. The mechanism(s) of formation of stable ds rAAV genomes from input ss DNA molecules has not been delineated, although second-strand synthesis and genome amplification by a rolling-circle model has been proposed. To begin to delineate a mechanism, we produced rAAV vectors in the presence of bacterial PaeR7 or Dam methyltransferase or constructed rAAV vectors labeled with different restriction enzyme recognition sites and introduced them into mouse hepatocytes in vivo. A series of molecular analyses demonstrated that second-strand synthesis and rolling-circle replication did not appear to be the major processes involved in the formation of stable ds rAAV genomes. Rather, recruitment of complementary plus and minus ss genomes and subsequent random head-to-head, head-to-tail, and tail-to-tail intermolecular joining were primarily responsible for the formation of ds vector genomes. These findings contrast with the previously described mechanism(s) of transduction based on in vitro studies. Understanding the mechanistic process responsible for vector transduction may allow the development of new strategies for improving rAAV-mediated gene transfer in vivo.

Occurrence of friabilin, a low molecular weight protein associated with grain softness, on starch granules isolated from some wheats and related species

Proteins of apparent M r , 14–15k by SDS—PAGE were identified on starches washed by aqueous procedures from soft endosperms of diploid Triticum and Aegilops spp. (AA, BB, DD and SS genomes). These proteins were analogous to the previously reported friabilins of hexaploid wheats (AABBDD genomes) and diploid rye ( Secale cereale , RR genome) and gave clear positive reactions in an ELISA test based on an antibody to soft wheat ( T. aestivun ) friabilin. Little or no friabilin-like protein was found on starches from hard endosperms of tetraploid wheats (AABB and AAGG genomes), diploid barley (HH genome) or Aegilops umbellulata (UU genome). It is proposed that these proteins should be designated friabilins-A, -B, -D, -S and -R, respectively, to identify the genome carrying the controlling genes.

Wheat germ agglutinin accumulation in coleoptiles of different genotypes of wheat. Localization by monoclonal antibodies

We have produced a library of 18 monoclonal antibodies (mABs) against wheat germ agglutinin (WGA). It was difficult to establish antibody-producing hybridomas when soluble WGA was used for immunization. The frequency of specific hybridomas was increased, however, by injecting mice with insoluble antigen-antibody complex.We distinguished groups of mABs that are especially efficient for particular immunoassays. One group (mABs 005, 006, 007, 009, 011, 014, 015, 016, 017, 018, 019) strongly immunostains denatured antigen on electroblots of sodium dodecyl sulfate polyacrylamide gels. A second group (all mABs except 012) shows high activity for WGA when native protein is analyzed by enzyme-linked immunosorbent assay. The third group (mABs 002, 005, 008, 009, 010, 011, 014, 016, 018, 019) works well for immunocytochemistry.We used the mABs to localize WGA in wheat varieties of various ploidy and with different ancestral wheat genomes. Whereas lectin is detected in the coleoptile of varieties with hexaploid and DD and SS genomes, WGA is absent in the coleoptile of the diploid Triticum monococcum (AA). Lectin accumulates in the coleoptile of mature embryos of T. monococcum, however, when they are treated with abscisic acid.