Showing papers on "Complementary DNA published in 2016"

Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons

Abstract: A protocol is described for sequencing the transcriptome of a cell nucleus. Nuclei are isolated from specimens and sorted by FACS, cDNA libraries are constructed and RNA-seq is performed, followed by data analysis. Some steps follow published methods (Smart-seq2 for cDNA synthesis and Nextera XT barcoded library preparation) and are not described in detail here. Previous single-cell approaches for RNA-seq from tissues include cell dissociation using protease treatment at 30 °C, which is known to alter the transcriptome. We isolate nuclei at 4 °C from tissue homogenates, which cause minimal damage. Nuclear transcriptomes can be obtained from postmortem human brain tissue stored at -80 °C, making brain archives accessible for RNA-seq from individual neurons. The method also allows investigation of biological features unique to nuclei, such as enrichment of certain transcripts and precursors of some noncoding RNAs. By following this procedure, it takes about 4 d to construct cDNA libraries that are ready for sequencing.

Identification of a phorbol ester-repressible v-src-inducible gene (immediate-early genes/Rous sarcoma virus/protein kinase C/chicken embryo fibroblasts)

Abstract: Chicken embryo fibroblasts (CEF) infected with a temperature-sensitive Rous sarcoma virus (RSV) mu- tant, tsNY72-4, express a set of pp6vWc-induced RNAs soon after shift to the permissive temperature. By subtractive and differential screening, we have cloned 12 of these sequences, 2 of which were c-fos and krox-24. Serum induced all the v-src-inducible genes tested, suggesting that these genes serve roles in normal cell division and are not specific to transfor- mation per se. Significantly, however, v-src produced pro- longed, and in some cases kinetically complex, patterns of Induction compared to serum. For most of the clones, phorbol 12-tetradecanoate 13-acetate (TPA) induced mRNAs with ki- netics similar to that of serum. However, one clone (CEF-4) was expressed in a biphasic manner. Another (CEF-10) was re- pressed by TPA at 1 hr, after which this mRNA was perma- nently induced. The pattern of repression-induction of CEF-10 mRNA is the inverse of protein kinase C (PKC) activity in the cell, suggesting that PKC actively represses this gene. In vivo expression of CEF-10 mRNA is restricted predominantly to the lung. A full-length CEF-10 cDNA encodes a 41-kDa protein that has an amino-terminal signal peptide for secretion, con- tains a markedly high number of cysteine residues, and shows no sequence similarity to known proteins.

cDNA cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts (in vitro transcription/sequence variation/population homology)

Abstract: The entire genome of tobacco mosaic virus (TMV) was copied into a series of subgenomic cDNA clones. cDNA sequences of the 5' and 3' ends of TMV were cloned separately. A synthetic oligonucleotide primer was used to generate a Pst I site at the 5' terminus, whereas a different primer was used to generate an Nde I site at the 3' terminus. This strategy permitted removal of non-TMV sequences from cloned cDNA inserts by treatment with exonuclease VII fol- lowing restriction endonuclease cleavage. Pst I linkers were added to TMV 3' terminal cDNAs. Subgenomic cDNA frag- ments were ligated together into several independent full- genomic constructions from which TMV cDNA sequences could be cleanly excised as a single fragment by Pst I digestion. Full-genomic TMV cDNA was ligated Immediately down. stream from the A phage promoter from pPMi and transcribed in vitro with Escherichia coli RNA polymerase. RNA tran- scripts from three of four full-genomic cDNA constructions were infectious, even though they contained 6 non-TMV nucleotides at the 3' end. Transcripts from a construction with 6 extra nucleotides at the 5' end also were infectious. Progeny virus from plants infected with cDNA transcripts appeared identical to the parental virus. Restriction maps of independent cDNA clones of the same regions of the genome were identical to each other and as predicted from the reported nucleotide sequence of TMV. Also, sequences of the 200 nucleotides proximal to the 5' ternini of four independent eDNA clones were identical to each other and to published sequences, suggesting that independent isolates of TMV may have remark- ably similar sequences.

Multiplexed, targeted profiling of single-cell proteomes and transcriptomes in a single reaction

Abstract: We present a scalable, integrated strategy for coupled protein and RNA detection from single cells. Our approach leverages the DNA polymerase activity of reverse transcriptase to simultaneously perform proximity extension assays and complementary DNA synthesis in the same reaction. Using the Fluidigm C1™ system, we profile the transcriptomic and proteomic response of a human breast adenocarcinoma cell line to a chemical perturbation, benchmarking against in situ hybridizations and immunofluorescence staining, as well as recombinant proteins, ERCC Spike-Ins, and population lysate dilutions. Through supervised and unsupervised analyses, we demonstrate synergies enabled by simultaneous measurement of single-cell protein and RNA abundances. Collectively, our generalizable approach highlights the potential for molecular metadata to inform highly-multiplexed single-cell analyses.

Hormone-responsive expression of an endogenous proviral gene of mouse mammary tumor virus after molecular cloning and gene transfer into cultured cells (glucocorticoid induction/GR mouse DNA library/A phage-MMTV recombinant clone/thymidine kinase gene cotransfection)

Abstract: A recombinant A phage containing mouse mam- mary tumor virus (MMTV) proviral DNA was isolated from a gene library constructed from GR mouse liver DNA. Restriction en- zyme analyses reveal that the cloned molecule contains a copy of one of the GR endogenous MMTV proviruses flanked on both sides by 2-3 kb of mouse genomic DNA. In this report we have examined the expression of the cloned MMTV provirus after cotransfection with the herpes thymidine kinase (TK; ATP:thymidine 5'-phos- photransferase, EC 2.7.1.21) gene and integration into mouse LTK- cells. Nine individual TK+ transformants were selected, and all were found to contain MMTV-transfected DNA. One of the TK+ transformants was chosen for further study. Total poly(A)-containing RNA was isolated from the cells, and liquid hybridization analyses with MMTV cDNA showed that it con- tained 0.02% MMTV-specific RNA. The sizes of the MMTV-spe- cific species were determined and found to correspond to the 35S and 24S mRNAs synthesized in MMTV-infected cells. Glucocor- ticoid hormones have been shown to increase the concentration of MMTV RNA in virus-infected cultured cells. Therefore, we tested the effect of dexamethasone on the concentration of MMTV- specific RNA in cells transfected with the MMTV proviral DNA. The amount of MMTV-specific poly(A)-containing RNA found in the cells grown in the presence of hormone was 0.17%. Therefore, dexamethasone causes an 8-fold increase in the amount of MMTV- specific RNA in mouse cells containing several copies of a cloned and transfected MMTV proviral gene.

cDNA cloning of a human mRNA preferentially expressed in hematopoietic cells and with homology to a GDP-dissociation inhibitor for the rho GTP-binding proteins (subtractive libraries/hematopoietic cDNA/guanine nucleotide exchange/differentiation/embryonic stem cell)

Abstract: We have identified the mRNA for a human gene, denoted D4, which is expressed at very high levels in hematopoietic cell lines and in normal cells of lymphoid and myeloid origin. The 1.5-kb transcript is absent or detectable only at low levels in nonhematopoietic tissues. D4 encodes a 201-amino acid protein with homology to rhoGDI, an inhibitor of GDP dissociation for the ras-homologous protein rho. D4 might function also as a regulator of guanine nucleotide exchange for small GTP-binding proteins. A homologous tran- script of similar size is also preferentially expressed in murine hematopoietic tissues. When totipotent murine embryonic stem cells develop in vitro into hematopoietic cells, the gene is activated with the onset of hematopoiesis. When hematopoietic cell lines are induced to differentiate, the expression of D4 is modulated. Thus, D4 appears to be a developmentally regu- lated gene. Its preferential expression in hematopoietic cells indicates that D4 likely plays some significant role in the growth and differentiation processes of hematopoietic cells. This sig- nificance is underscored by increasing evidence for the involve- ment of regulators of G proteins in clinical diseases.

Synthetic evolutionary origin of a proofreading reverse transcriptase

Abstract: Most reverse transcriptase (RT) enzymes belong to a single protein family of ancient evolutionary origin. These polymerases are inherently error prone, owing to their lack of a proofreading (3′- 5′ exonuclease) domain. To determine if the lack of proofreading is a historical coincidence or a functional limitation of reverse transcription, we attempted to evolve a high-fidelity, thermostable DNA polymerase to use RNA templates efficiently. The evolutionarily distinct reverse transcription xenopolymerase (RTX) actively proofreads on DNA and RNA templates, which greatly improves RT fidelity. In addition, RTX enables applications such as single-enzyme reverse transcription–polymerase chain reaction and direct RNA sequencing without complementary DNA isolation. The creation of RTX confirms that proofreading is compatible with reverse transcription.

Characterization of mouse myelin basic protein messenger RNAs with a myelin basic protein cDNA clone (multiple mRNAs/RNA blot analysis/cDNA cloning)

Abstract: Using a family of synthetic tetradecamer oli- gonucleotides as a primer for cDNA synthesis and a second family of tetradecamers as a hybridization probe, we have pre- pared and isolated a cDNA clone of mouse myelin basic protein (MBP). The clone, pNZ111, corresponds to the region of the mRNA that codes for an amino acid sequence present in all four major forms of MBP. The relative abundance of MBP mRNA, estimated by dot blot hybridization, increased with the age of the mouse to a maximum at 18 days, then decreased to about one-fourth of that amount at later ages. Mouse MBP mRNAs, selected by their ability to hybridize to the clone, translate into the four forms of myelin basic protein. In RNA blot analyses, pNZ1ll hybridized to multiple species of mouse mRNA. The predominant hybridization is to a broad band of RNAs ranging in length from 2,350 to 2,100 bases. These mRNA species are extremely long, considering that the largest MBP could be encoded by approximately 600 bases. In addi- tion to these, there are also minor bands that hybridize with pNZ1ll, including a band of 4,100 bases and smaller ones of 1,900, 1,500, and 1,200 bases. same relationship to the 14-kDa MBP (13). Myelination occurs postnatally in mouse brain beginning 8-10 days postpartum and continuing actively for 7-10 wk, with the maximal rate of myelin deposition occurring at about 18 days (4). Maximal synthesis of the 18.5-kDa and 14- kDa MBPs occurs at 18 days in vivo and coincides closely with the peak of myelin synthesis (14). During myelin matu- ration in the mouse, the proportions of the four MBPs in the membrane change (15-17) and this may be due to alterations in the relative rates of synthesis of the four proteins with age. With maturation, the proportion of the two minor MBPs (i.e., the 21.5 kDa and 17 kDa in myelin falls relative to the 18.5-kDa proteins and the 14-kDa/18.5-kDa MBP ratio in- creases dramatically. This latter change has been correlated with the relative rate of synthesis of these two proteins in vivo (14). Recently, Carson et al. (18) have identified trace amounts of larger forms of MBP that appear during the early stages of myelination. Identification of these larger forms has depend- ed on immunoreagents and the relationships of the amino acid sequences to the major MBPs have, as yet, not been determined. We have recently initiated a study of the component mo- lecular events that culminate in the formation of compact myelin. The initial focus of this study is the interrelation- ships of the four MBPs and the developmental program that controls their synthesis and accumulation into myelin. In the present communication, we report the isolation of a cDNA clone of MBP mRNA by using synthesized families of tetra- decamer oligodeoxyribonucleotides as primers for cDNA synthesis and as probes to identify the desired clone. The cDNA clone specifies a sequence found in the four predomi- nant MBPs and has been used to analyze the complexity, size, and cellular distribution of MBP mRNAs.

Characterization of the chicken vimentin gene: Single copy gene producing multiple mRNAs (cytoskeleton/intermediate filament proteins/muscle/termination/polyadenylylation)

Abstract: Genomic clones and cDNA plasmids were iso- lated for the intermediate filament protein vimentin from chicken. The identity of the various clones was determined both by mRNA selection (Paterson, B. M. & Roberts, B. E. (1981) in Gene Am- plification and Analysis, Structural Analysis or Nucleic Acids, eds. Chirikjian, J. G. & Papas, T. S. (Elsevier, North Holland), Vol. 2, pp. 418-435) and nucleotide sequence analysis. Restriction analysis, hybridization data, and heteroduplex studies confirmed that all of the genomic isolates contained overlapping fragments of an identical vimentin gene. No evidence for the existence of a second vimentin gene could be found by a Southern analysis either by using coding fragments from the purified vimentin gene or by using cDNA plasmids as probe. Likewise, copy-number experi- ments verified that the vimentin gene was present only once in the haploid chicken genome. However, in a RNA blot analysis, at least two equally abundant vimentin mRNA species of approximately 2,200 and 2,500 nucleotides in length were detected in all RNAs tested. Sequence analysis revealed that the vimentin gene con- tained two sets of tandem polyadenylylation sites, 249 and 532 nucleotides downstream from the stop codon for protein synthesis. It is proposed that the larger mRNA species arise because of com- plete transcription of the 3'-end of the vimentin gene (560 nu- cleotides of 3' nontranslated sequence), whereas the smaller

Sensitive and specific miRNA detection method using SplintR Ligase

Abstract: We describe a simple, specific and sensitive microRNA (miRNA) detection method that utilizes Chlorella virus DNA ligase (SplintR(®) Ligase). This two-step method involves ligation of adjacent DNA oligonucleotides hybridized to a miRNA followed by real-time quantitative PCR (qPCR). SplintR Ligase is 100X faster than either T4 DNA Ligase or T4 RNA Ligase 2 for RNA splinted DNA ligation. Only a 4-6 bp overlap between a DNA probe and miRNA was required for efficient ligation by SplintR Ligase. This property allows more flexibility in designing miRNA-specific ligation probes than methods that use reverse transcriptase for cDNA synthesis of miRNA. The qPCR SplintR ligation assay is sensitive; it can detect a few thousand molecules of miR-122. For miR-122 detection the SplintR qPCR assay, using a FAM labeled double quenched DNA probe, was at least 40× more sensitive than the TaqMan assay. The SplintR method, when coupled with NextGen sequencing, allowed multiplex detection of miRNAs from brain, kidney, testis and liver. The SplintR qPCR assay is specific; individual let-7 miRNAs that differ by one nucleotide are detected. The rapid kinetics and ability to ligate DNA probes hybridized to RNA with short complementary sequences makes SplintR Ligase a useful enzyme for miRNA detection.

Cloning of a human complement component C4 gene (HLA class III antigen/cosmid library/synthetic oligonucleotide/cDNA library)

Abstract: Six overlapping cosmid clones having an average insert size of 40 kilobase pairs were identified and isolated from a human genomic library by using a cDNA probe, Alu-7, specific for the amino acid sequence of C4d, a known region of the fourth component of human complement. Analysis of these genomic clones by restriction digestion and Southern blotting shows that all six probably contain the same complete C4 gene. Nucleotide .sequence comparison of the genomic clone Cos-A and the cDNA clone Alu-7 shows an identical sequence except for the presence of a 1,500-base,pair intron in the genomic sequence. The amino acid sequence predicted from the nucleotide sequence agrees with

Isolation of genomic DNA clones spanning the entire fibronectin gene (recombinant DNA/intervening sequence/gene duplication/evolution/cell adhesion)

Abstract: Overlapping recombinant clones that appear to encompass the entire fibronectin gene have been isolated by step- wise screening of a library of chicken genomic DNA fragments. The first genomic clone was isolated by using a cloned fibronectin cDNA hybridization probe. The remaining clones were obtained by using defined fragments of this and successive genomic clones as probes. Their relationships and overlaps were determined by electron microscopy, restriction mapping, and heteroduplex anal- ysis. Based on electron microscopic analysis of hybrids between these clones and fibronectin mRNA, the gene is approximately 48 kilobases long, more than 5 times larger than the corresponding mRNA. This large gene contains at least 48 exons interrupted by introns of highly variable size. The total exon size as estimated by R-loop analysis is 8 kilobases, similar to the mRNA for fibronectin. With the exception of the 3'- and 5'-terminal exons, the exons are small and roughly similar in size. The. average exon size is 147 ? 37 base pairs, corresponding to a protein unit of 50 amino acids. The nucleotide sequence.of one of these exons was determined. The deduced amino acid sequence has marked homologies with one type of repetitive protein sequence unit known to exist in bo- vine fibronectin. These results suggest that the gene for fibro- nectin may-have arisen by multiple gene duplications of a pri- mordial gene or genes 150 base pairs long.

Transgenic poplar overexpressing the endogenous transcription factor ERF76 gene improves salinity tolerance.

Abstract: The ethylene response factor (ERF) family is one of the largest plant-specific transcription factor families, playing an important role in plant development and response to stresses. The ERF76 gene is a member of the poplar ERF transcription factor gene family. First, we validated that the ERF76 gene expressed in leaf and root tissues is responsive to salinity stress. We then successfully cloned the ERF76 cDNA fragment containing an open reading frame from di-haploid Populus simonii × Populus nigra and proved that ERF76 protein is targeted to the nucleus. Finally, we transferred the gene into the same poplar clone by the Agrobacterium-mediated leaf disc method. Using both RNA-Seq and reverse transcription-quantitative polymerase chain reaction, we validated that expression level of ERF76 is significantly higher in transgenic plants than that in the nontransgenic control. Using RNA-Seq data, we have identified 375 genes that are differentially expressed between the transgenic plants and the control under salt treatment. Among the differentially expressed genes, 16 are transcription factor genes and 45 are stress-related genes, both of which are upregulated significantly in transgenic plants, compared with the control. Under salt stress, the transgenic plants showed significant increases in plant height, root length, fresh weight, and abscisic acid (ABA) and gibberellin (GA) concentration compared with the control, suggesting that overexpression of ERF76 in transgenic poplar upregulated the expression of stress-related genes and increased the ability of ABA and GA biosynthesis, which resulted in stronger tolerance to salt stress.

Two ornithine decarboxylase mRNA species in mouse kidney arise from size heterogeneity at their 3' termini (androgen action/ornithine decarboxylase genes/polyadenylylation/blot hybridization/DNA sequencing)

Abstract: Ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17) mRNA present In mouse kidney comprises two species with molecular sizes of =2.2 and -=2.7 kilobases (kb). cDNA clones prepared from murine kidney OrnDCase mRNA were used to determine the reason for the size heterogeneity of these mRNAs. Two of the cDNA clones (pODC16 and pODC74) that differed at the 3' termini were isolated and sequenced. DNA sequencing indicated that each cDNA had a poly(A) tail; however, pODC74 was 429 nucleotides longer than pODC16 at the 3' end and contained two AATAAA signals for poly(A) addition. That the longer cDNA corresponded to the larger mRNA was confirmed by hybridization of a unique Pst I/Pst I fragment from the 3' terminus of pODC74 only to the 2.7-kb OrnDCase mRNA. The two cDNAs did not represent fulllength copies of OrnDCase mRNAs and were 1199 (pODC16) and 1204 base pairs (bp) (pODC74) long. There were five mismatches in their 759-bplong overlapping nucleotide sequence, suggesting that the 2.2and 2.7-kb OrnDCase mRNAs may be products of two separate, yet very similar, OrnDCase genes. Androgen regulation of the accumulation of these two OrnDCase mRNAs appeared to occur coordinately, as testosterone administration brought about comparable increases in their concentrations in mouse kidney. Ornithine decarboxylase (OmDCase; L-ormithine carboxylyase, EC 4.1.1.17) catalyzes the conversion of ornithine to putrescine and is the first and apparently rate-controlling enzyme in polyamine biosynthesis (1-3). In the murine kidney, OrnDCase activity and the enzyme protein concentration are increased by androgen treatment with relatively rapid kinetics, with a maximal induction at 18-24 hr after steroid administration (4, 5). In addition to enhancing enzyme synthesis, androgen administration increases OrnDCase concentration through prolonging the biological half-life of the enzyme protein (5, 6). To better understand the mechanisms regulating OmDCase synthesis, workers in our own (7) and other laboratories (8-10) have prepared cDNA clones for the mRNA encoding OmDCase. When these cDNAs were used to identify OrnDCase mRNA by hybridization after agarose gel electrophoresis, two mRNA species of =2.2 and =2.7 kilobases (kb) were found (7, 10). The nucleotide sequence and deduced amino acid sequence of the shorter OrnDCase mRNA have been recently published (11, 12). We report here the nucleotide sequence of the 3' ends of the two OrnDCase mRNA species in mouse kidney. From the sequences and from RNA blot hybridization data using the unique 3'terminal probe from one of the clones, we conclude that the size heterogeneity of the two OrnDCase mRNAs is due to the dissimilar lengths of their 3' noncoding regions. In addition, The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. ?1734 solely to indicate this fact. DNA sequence data suggest that they could be products of two similar but separate OrnDCase genes. MATERIALS AND METHODS Animnals. Mature male and female NCS [randomly bred strain Rku:NCS(s) SPF] and 129/J mice were from The Rockefeller University and The Jackson Laboratory, respectively. The animals were treated for 7 days with testosteronecontaining Silastic implants releasing either 40 or 200 .tg of testosterone per day. OrnDCase cDNAs. The preparation and cloning of the two OrnDCase eDNAs (pODC16 and pODC74) have been described (7, 13). The plasmids were propagated in Escherichia coli strain LE 392 and purified by a modification of the method of Ish-Horowicz and Burke (14) followed by CsCl density gradient centrifugation. End-Labeling and Sequencing of cDNAs. Plasmids carrying OrnDCase cDNAs were digested overnight with the appropriate restriction enzymes, ethanol-precipitated, and labeled at the 3' ends with [a-32P]dNTPs using the Klenow fragment of DNA polymerase I, or by the terminal deoxyribonucleotidyltransferase-catalyzed addition of cordycepin 5'-[a32P]triphosphate. Labeled fragments were isolated by electrophoresis on 6% polyacrylamide gels under non-denaturing conditions, recovered from the gel slices by electroelution, and ethanol-precipitated. Isolated fragments were recut, purified by electrophoresis, and sequenced by the chemical degradation method of Maxam and Gilbert (15), as modified by Catterall et al. (16). Each sample was subjected to electrophoresis on 20%, two 10% and 8% polyacrylamide sequencing gels with 7 M urea/90 mM Tris-HCl/90 mM borate/2 mM EDTA buffer, pH 8.3, and autoradiographed at -70?C using Kodak XAR-5 fim. Isolation of Renal RNA and Gel Blot Hybridizations. Total RNA was isolated from murine kidney by the lithium chloride/urea method (17) and enriched for poly(A)-containing RNA by oligo(dT)-cellulose chromatography (18). RNA samples (4-6 ug) were fractionated on 1% agarose gels containing 2.2 M formaldehyde (19), transferred to nitrocellulose filters, and hybridized with radioactive probes (20). The probes used for hybridization were isolated from pODC74 or pODC16 by preparative gel electrophoresis on 6% polyacrylamide gels after cleavage with appropriate restriction enzymes, and labeled with [a-32P]dCTP by nicktranslation. The terminal Pst I/Pst I fragment of pODC74 (from the cloning site to the first Pst I site at the 3' end) was the unique 3' probe, whereas the Hpa II/Hha I fragment of pODC74 or the 5' Pst I/Pst I fragment of pODC16 were the probes representing overlapping sequences (see Fig. 1). Quantitation of the signals was achieved by excision of the Abbreviations. OrnDCase, ornithine decarboxylase; kb, kilobase(s); bp, base pair(s). *To whom reprint requests should be addressed.

Characterization of a Novel Polerovirus Infecting Maize in China.

Abstract: A novel virus, tentatively named Maize Yellow Mosaic Virus (MaYMV), was identified from the field-grown maize plants showing yellow mosaic symptoms on the leaves collected from the Yunnan Province of China by the deep sequencing of small RNAs. The complete 5642 nucleotide (nt)-long genome of the MaYMV shared the highest nucleotide sequence identity (73%) to Maize Yellow Dwarf Virus-RMV. Sequence comparisons and phylogenetic analyses suggested that MaYMV represents a new member of the genus Polerovirus in the family Luteoviridae. Furthermore, the P0 protein encoded by MaYMV was demonstrated to inhibit both local and systemic RNA silencing by co-infiltration assays using transgenic Nicotiana benthamiana line 16c carrying the GFP reporter gene, which further supported the identification of a new polerovirus. The biologically-active cDNA clone of MaYMV was generated by inserting the full-length cDNA of MaYMV into the binary vector pCB301. RT-PCR and Northern blot analyses showed that this clone was systemically infectious upon agro-inoculation into N. benthamiana. Subsequently, 13 different isolates of MaYMV from field-grown maize plants in different geographical locations of Yunnan and Guizhou provinces of China were sequenced. Analyses of their molecular variation indicate that the 3′ half of P3–P5 read-through protein coding region was the most variable, whereas the coat protein- (CP-) and movement protein- (MP-)coding regions were the most conserved.

Isolation of proteins associated with kinetoplast DNA networks in vivo (DNA-binding protein/DNA-protein crosslinking/mitochondrial DNA/presequence)

Abstract: Kinetoplast DNA (kDNA), the mitochondrial DNA of trypanosomes, is a highly condensed disc-shaped network of catenated DNA circles consisting of maxicircles, the equivalent of conventional mitochondrial DNA, and several thousand smaller circular DNAs termed minicircles. Upon cell lysis, kDNA expands, giving rise to a two-dimensional network of catenated circles with an overall diameter close to that of the whole cell. To identify proteins associated with the condensed form of kDNA in the cell, proteins were reversibly crosslinked to kDNA in whole cells of Crithidiafasciculata by formaldehyde treatment. Crosslinked networks were purified and found to retain a condensed structure which becomes fully expanded upon proteinase K treatment or reversal of the crosslinks by heating at 65?C. Five low molecular weight proteins released from the kDNA by heat treatment were purified by polyacryl- amide gel electrophoresis and their amino-terminal sequences were determined. PCR amplification and sequence analysis of cDNA sequences between these amino-terminal sequences and the miniexon (spliced leader) sequence present at the 5' end of all C. fasciculata mRNAs predicts the presence of 9-amino acid presequences with features characteristic of mitochondrial presequences on three of the proteins. Two of these proteins are lysine-rich basic proteins. These fmdings suggest that basic proteins may play a role in the condensation of kDNA in the kinetoplast and that these proteins are imported into the kinetoplast by a mechanism involving a cleavable presequence.

Primary structures of bovine liver low molecular weight kininogen precursors and their two mRNAs (bradykinin/recombinant DNA/DNA sequence/internal homology/RNA processing)

Abstract: By using a mixture of synthetic oligodeoxyribonu- cleotides as a probe, cloned cDNA sequences specific for low mo- lecular weight (LMW) kininogen have been isolated from a cDNA library of bovine liver mRNA sequences. Nucleotide sequence analyses of cloned cDNA inserts have revealed that bovine liver LMW kininogens are encoded by at least two very similar but dis- tinct mRNAs. The corresponding amino acid sequences show that the LMW kininogen precursors of the two types, composed of 436 and 434 amino acid residues, both contain two internally homol- ogous sequences in the amino-terminal portion between a signal peptide and a bradykinin moiety. The two mRNAs exhibit 15 nu- cleotide substitutions and 6 nucleotide deletions/additions in their protein-coding regions. The replacement of 13 amino acid residues and the deletions/additions of 2 amino acid residues in the two LMW kininogen precursors are all localized within the internally homologous regions, implying that these regions may be biologi- cally significant in relation to the existence of two LMW kinino- gens. The nucleotide changes in the two mRNAs also occur in the limited portions that principally encode the internally homologous amino acid sequences. This suggests that the mRNAs are tran- A number of questions regarding the structures, functions, and biosynthesis of kininogens remain to be elucidated. The construction of bacterial plasmids containing the mRNA se- quences for kininogens is a direct approach to analyzing the amino acid sequences, the mRNAs, and the genes for these proteins. We report here the cloning of DNA sequences com- plementary to the bovine liver mRNAs coding for LMW kinin- ogens. Nucleotide sequence analyses of cloned cDNA inserts have revealed that bovine LMW kininogens are encoded by at least two very similar but distinct mRNAs. The intriguing pri- mary structures of the LMW kininogen precursors and the char- acteristic nucleotide sequence relationship between the two mRNAs are reported.

Unusual features in the nucleotide sequence of a cDNA clone derived from the common region of avian sarcoma virus

Abstract: We have constructed a recombinant plasmid containing a 700-base pair (bp) cDNA copy of the common re- gion present at the 3' end of Schmidt-Ruppin avian sarcoma virus (ASV) 21S mRNA. The cDNA was inserted into plasmid pBR322 at the Pst I site by the G-C tailing method. A restriction map of the cloned insert from a recombinant plasmid pSRI in- dicates that it corresponds to the 3' end of the ASV genome. R-loop analysis with ASV genomic RNA indicates that the insert is colinear with the ASV genome over most of its length. The sequence of 331 bp at the 3' end of the DNA insert was deter- mined and shows that the insert contains extra sequences not found at the 3' end of ASV genomic RNA. Following the termi- nally redundant sequence of 20 bp that has been found at the extreme 3' end of genomic RNA is a sequence of 79 bp that is almost identical to that located immediately next to the 20-bp repeat at the 5' end of ASV genomic RNA. This is followed by 18 bp of unique sequence, possibly of host origin. The structure of the clone suggests that ASV mRNA may differ from genomic RNA at its 3' end and that 21S mRNA is transcribed from inte- grated ASV DNA and contains at its 3' end sequences derived both from the 5' end of the ASV genome and from host DNA adjacent to the site of integration. The presence of termination codons in all three reading frames suggests that the common region probably does not contain coding sequences. However, the presence of sequences that resemble probable promoter sites supports the possibility that this region may be involved in the regulation of transcription.

cDNA Library Enrichment of Full Length Transcripts for SMRT Long Read Sequencing.

Abstract: The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci.

Identification, cloning and characterization of R2R3-MYB gene family in canola (Brassica napus L.) identify a novel member modulating ROS accumulation and hypersensitive-like cell death

Abstract: The R2R3-MYB proteins comprise one of the largest families of transcription factors in plants. Although genome-wide analysis of this family has been carried out in some plant species, little is known about R2R3-MYB genes in canola (Brassica napus L.). In this study, we have identified 76 R2R3-MYB genes in the canola genome through mining of expressed sequence tags (ESTs). The cDNA sequences of 44 MYB genes were successfully cloned. The transcriptional activities of BnaMYB proteins encoded by these genes were assayed in yeast. The subcellular localizations of representative R2R3-MYB proteins were investigated through GFP fusion. Besides, the transcript abundance level analysis during abiotic conditions and ABA treatment identified a group of R2R3-MYB genes that responded to one or more treatments. Furthermore, we identified a previously functionally unknown MYB gene-BnaMYB78, which modulates reactive oxygen species (ROS)-dependent cell death in Nicotiana benthamiana, through regulating the transcription of a few ROS- and defence-related genes. Taken together, this study has provided a solid foundation for understanding the roles and regulatory mechanism of canola R2R3-MYB genes.

Reverse Transcription of Threose Nucleic Acid by a Naturally Occurring DNA Polymerase

Abstract: Recent advances in polymerase engineering have enabled the replication of xenonucleic acid (XNA) polymers with backbone structures distinct from those found in nature. By introducing a selective amplification step into the replication cycle, functional XNA molecules have been isolated by in vitro selection with binding and catalytic activity. Despite these successes, coding and decoding genetic information in XNA polymers remains limited by the fidelity and catalytic efficiency of engineered XNA polymerases. In particular, the process of reverse transcribing XNA back into DNA for amplification by PCR has been problematic. Here, we show that Geobacillus stearothermophilus (Bst) DNA polymerase I functions as an efficient and faithful threose nucleic acid (TNA)-dependent DNA polymerase. Bst DNA polymerase generates ∼twofold more cDNA with threefold fewer mutations than Superscript II (SSII), which was previously the best TNA reverse transcriptase. Notably, Bst also functions under standard magnesium-dependent conditions, whereas SSII requires manganese ions to relax the enzyme's substrate specificity. We further demonstrate that Bst DNA polymerase can support the in vitro selection of TNA aptamers by evolving a TNA aptamer to human α-thrombin.

Selective amplification of an mRNA and related pseudogene for a human ADP-ribosylation factor, a guanine nucleotide-dependent protein activator of cholera toxin (guanine nucleotide-binding proteins/adenylate cyclase/polymerase chain reaction)

Abstract: ADP-ribosylation factors (ARFs) are -20- kDa proteins that act as GTP-dependent allosteric activators of cholera toxin. With deoxyinosine-containing degenerate oligo- nucleotide primers corresponding to conserved GTP-binding domains in ARFs, the polymerase chain reaction (PCR) was used to amplify simultaneously from human DNA portions of three ARF genes that include codons for 102 amino acids, with intervening sequences. Amplification products that differed in size because of differences in intron sizes were separated by agarose gel electrophoresis. One amplified DNA contained no introns and had a sequence different from those of known ARFs. Based on this sequence, selective oligonucleotide probes were prepared and used to isolate clone IARF 4, a putative ARF pseudogene, from a human genomic library in A phage EMBL3. Reverse transcription-PCR was then used to clone from human poly(A)+ RNA the cDNA corresponding to the expressed homolog of PARF 4, referred to as human ARF 4. It appears that IARF 4 arose during human evolution by integration of processed ARF 4 mRNA into the genome. Human ARF 4 differs from previously identified mammalian ARFs 1, 2, and 3. Hybridization of ARF 4-specific oligonucle- otide probes with human, bovine, and rat RNA revealed a single 1.8-kilobase mRNA, which was clearly distinguished from the 1.9-kilobase mRNA for ARF 1 in these tissues. The PCR provides a powerful tool for investigating diversity in this and other multigene families, especially with primers targeted at domains believed to have functional significance.

Enrichment by hybridisation of long DNA fragments for Nanopore sequencing.

Abstract: Enrichment of DNA by hybridisation is an important tool which enables users to gather target-focused next-generation sequence data in an economical fashion. Current in-solution methods capture short fragments of around 200–300 nt, potentially missing key structural information such as recombination or translocations often found in viral or bacterial pathogens. The increasing use of long-read third-generation sequencers requires methods and protocols to be adapted for their specific requirements. Here, we present a variation of the traditional bait–capture approach which can selectively enrich large fragments of DNA or cDNA from specific bacterial and viral pathogens, for sequencing on long-read sequencers. We enriched cDNA from cultured influenza virus A, human cytomegalovirus (HCMV) and genomic DNA from two strains of Mycobacterium tuberculosis (M. tb) from a background of cell line or spiked human DNA. We sequenced the enriched samples on the Oxford Nanopore MinION™ and the Illumina MiSeq platform and present an evaluation of the method, together with analysis of the sequence data. We found that unenriched influenza A and HCMV samples had no reads matching the target organism due to the high background of DNA from the cell line used to culture the pathogen. In contrast, enriched samples sequenced on the MinION™ platform had 57 % and 99 % best-quality on-target reads respectively.

Straightforward and sensitive RT-qPCR based gene expression analysis of FFPE samples

Abstract: Fragmented RNA from formalin-fixed paraffin-embedded (FFPE) tissue is a known obstacle to gene expression analysis. In this study, the impact of RNA integrity, gene-specific reverse transcription and targeted cDNA preamplification was quantified in terms of reverse transcription polymerase chain reaction (RT-qPCR) sensitivity by measuring 48 protein coding genes on eight duplicate cultured cancer cell pellet FFPE samples and twenty cancer tissue FFPE samples. More intact RNA modestly increased gene detection sensitivity by 1.6 fold (earlier detection by 0.7 PCR cycles, 95% CI = 0.593–0.850). Application of gene-specific priming instead of whole transcriptome priming during reverse transcription further improved RT-qPCR sensitivity by a considerable 4.0 fold increase (earlier detection by 2.0 PCR cycles, 95% CI = 1.73–2.32). Targeted cDNA preamplification resulted in the strongest increase of RT-qPCR sensitivity and enabled earlier detection by an average of 172.4 fold (7.43 PCR cycles, 95% CI = 6.83–7.05). We conclude that gene-specific reverse transcription and targeted cDNA preamplification are adequate methods for accurate and sensitive RT-qPCR based gene expression analysis of FFPE material. The presented methods do not involve expensive or complex procedures and can be easily implemented in any routine RT-qPCR practice.