The development of a novel vector for construction of a full-length cDNA library

A new original vector pEM-(dT)₄₀(f+) has been prepared. It can be used for cDNA library construction from polyadenylated mRNA, isolated from various sources. The vector pGEM-(dT)₄₀f(+) is initially transformed into single stranded and then into a linear form and its (dT)₄₀ tail at the 3′-end is used as the vector-primer for synthesis of the first strand cDNA. The use of a synthetic oligonucleotide complementary to the vector and recombinant DNA results in vector circularization and synthesis of the second strand cDNA. This approach has the following advantages: (1) it significantly simplifies cDNA library construction, which includes three steps; (2) full-length cDNA library construction is achieved by adding a (dC)_n homopolymer tail to the 5′end; (3) preparation of a clone library requires a few milligrams of total RNA; (4) it is possible to obtain cDNA clones up to 10 kbp; (5) it does not require PCR reaction (which can induce artifact mutations in cDNA sequences); (6) this approach does not employ restrictase treatment and chimeric cDNA products are not formed.     

Diversity,complexity and transmission of double-stranded RNA elements in Chalara elegans (synanam. Thielaviopsis basicola)

Double-stranded (ds) RNA banding patterns were determined in 21 wild-type strains of the soilborne plant pathogen Chalara elegans originating from different geographic regions worldwide. Five strains, each with a unique dsRNA pattern, were selected for cDNA cloning, northern blot analysis and dsRNA transmission experiments. Four strains contained multiple (up to 6) dsRNA elements (2.0 kbp to 12 kbp in size) and one strain contained a single 2.8 kbp fragment. These five strains were distinguished from one another by their unique RAPD-PCR patterns. Seven partial cDNA clones were derived from the predominant 2.8, 5.3, and 12 kbp dsRNA elements. Nucleotide sequence analysis and northern blot hybridizations revealed a high degree of genetic dissimilarity among the different molecular-size dsRNA elements, even those found within a single strain. Four clones from the 5.3 kbp dsRNA fragment showed a 23-43 % amino acid identity to either the coat protein or RNA-dependent RNA polymerase regions of viruses in the Totiviridae. One clone from the 2.8 kbp dsRNA fragment had a 55-57 % amino acid identity to the RdRp region of viruses in the Narnaviridae. Two clones from the 12 kbp dsRNA fragment showed no significant homology to any known virus group. Colonies derived from 100 single-conidia isolates of C. elegans strains with the 2.8, 5.3 and 12 kbp elements all contained the corresponding dsRNA element, indicating that dsRNA transmission through conidia was highly efficient, regardless of molecular size. However, transmission of dsRNA between the mycelium of strains of C. elegans could not be achieved in this study. Genetically unique strains carrying diverse dsRNA elements appear to have evolved within populations of C. elegans. Based on our findings, there are at least 3 groups of viruses present in C. elegans.     

Isolation of a genomal clone containing chicken histone genes.

We have used enriched chicken histone cDNA to select genomal clones from a chicken library. Because the cDNA probe also contained other sequences, a further screening of positive plagues with negative probes eliminated most non-histone gene clones. One 'positively-selected' genomal clone, lambda CH-01, hybridised with cloned sea-urchin histone genes and also detected histone genes in EcoRI-digested genomal sea-urchin DNA. Limited DNA sequencing of HaeIII fragments identified two sequences within the coding region of chicken histone H2A. A third fragment predicted an amino acid sequence with strong homology to an H1 histone sequence.     

Molecular Cloning of DNA Complementary to the RNA-Genome of Plum Pox Virus (PPV)

Genomic RNA of plum pox virus (PPV) was used as a template for the synthesis of complementary DNA (cDNA). The generated cDNA molecules were subsequently cloned into pBR 322. A physical map covering 9700 bases of the PPV genome was constructed from 8, clones by hybridization and restriction endonuclease digestion. Clone pPPV-NAT 309, starting at the 3′-end, with an 866 bp insert was used in Northern- and Dot-hybridizations for the detection of single-stranded viral RNA in total nucleic acid as well as in sap preparations of PPV infected Nicotiana clevelandii. The nucleotide sequence of this clone was determined, the amino acid sequence of the coat protein C-terminal part was deduced and compared with four other coat proteins of potyviruses.     

Location of DNA sequences complementary to small nuclear repeat RNA (fr 3-RNA) in relation to DNA sequences coding for albumin and α-fetoprotein in rat liver cells

Rat liver nuclei contain a 29-nucleotides-long RNA (fr 3-RNA) which is transcribed from middle repetitive DNA sequences. By Southern analysis of restriction fragments of rat albumin and α-fetoprotein genomic clones, DNA sequences complementary to this RNA were detected on a 4.6 kbp EcoRI fragment located 600 bp downstream from the termination exon of the albumin gene and on a 2 kbp EcoRI-HindIII fragment located 10 kbp downstream from the restriction fragment containing the α-fetoprotein site. No sequence complementary to this RNA was found either in the introns of exons of both genes or in the regions extending 7 kbp upstream from the first albumin exon and 10 kbp upstream of the first α-fetoprotein exon. We concluded that sequences complementary to fr 3-RNA are present at the 3′-end flanking regions of the rat albumin and α-fetoprotein gene complexes.     

Long internal inverted repeat in a yeast viral double-stranded RNA.

The Saccharomyces cerevisiae viruses are non-infectious double-stranded (ds) RNA viruses present in most laboratory strains of yeast. Their genome consists of one or more dsRNAs separately encapsidated in particles composed mainly of one polypeptide, which has a Mr of 88 kdaltons in the best-studied viral subtype. A large viral dsRNA (L1, of 4.7 kb) encodes the capsid polypeptide. We have determined the sequences of a number of cDNA clones homologous to portions of L1 and mapped them by a novel heteroduplex technique. Several of these clones originate from a region of L1 2.3-2.5 kb from the 5' end of the plus strand that contains stop codons in all three reading frames in the plus strand. We therefore suspect that the capsid polypeptide gene lies in the 5' 2.3-2.6 kb of the plus strand. One of the cloned cDNAs has an inverted repeat of 170 bp that appears to be present in its parental RNA. The inverted repeat in L1 is the longest known inverted repeat in a viral dsRNA and the only known non-terminal inverted repeat. It might serve the function of creating two mRNAs from one viral dsRNA.     

Human apolipoprotein A-II: complete nucleic acid sequence of preproapoA-II

The complete cDNA nucleic acid sequence of preproapolipoprotein (apo) A-II, a major protein constituent of high density lipoproteins, has been determined on clones from a human liver ds-cDNA library. Clones containing ds-cDNA for apoA-II were identified in the human liver ds-cDNA library using synthetic oligonucleotides as probes. Of 3200 clones screened, 4 reacted with the oligonucleotide probes. The DNA sequence coding for amino acids ?17 to +17 of apoA-II were determined by Maxam-Gilbert sequence analysis of restriction fragments isolated from one of these clones, pMDB2049. The remainder of the cDNA sequence was established by sequence analysis of a primer extension product synthesized utilizing a restriction fragment near the 5'-end of clone pMDB2049 as primer with total liver mRNA. The apoA-II mRNA encodes for a 100 amino acid protein, preproapoA-II that has an 18 amino acid prepeptide and a 5 amino acid propeptide terminating with a basic dipeptide (Arg-Arg) at the cleavage site to mature apoA-II.     

Comparative analysis of Xenopus tropicalis and Xenopus laevis vitellogenin gene sequences

Analysis of cDNA clones synthesized from vitellogenin mRNA of X. tropicalis revealed three different types of cDNA clones, i.e. A, A* and B. A and A* clones have a sequence divergence of about 6% and are both related to X. laevis vitellogenin cDNAs of subgroup A1 as well as A2 with a sequence divergence of 6-9%. B clones however, are related to X. laevis cDNA clones of subgroup B1 and B2 with a sequence divergence of about 7%. While the A and B clones correspond to vitellogenin mRNAs of similar abundance, A* clone is complementary to a vitellogenin mRNA about 100 fold less abundant than A and B mRNAs although all three vitellogenin mRNAs are encoded by single copy genes. Furthermore, two forms of A* mRNA were found. One of the two is lacking an internal fragment of about 900 bp. Since this DNA fragment is highly repeated in the genome, we suggest that this A* clone was synthesized from a processing intermediate of the A* precursor vitellogenin mRNA.     

Production of cloned cDNA from a Swedish barley yellow dwarf virus isolate

A cDNA library was produced from the RNA of a Swedish MAV-like isolate of barley yellow dwarf virus (BYDV). The procedure involved random priming and the ds cDNA was cloned into the EcoRl site of the plasmid pUC19. Among the clones obtained some hybridised specifically with MAV-like isolates whereas others also hybridised with PAV-like isolates. Only very weak hybridisation was observed with an RPV-like isolate. An Australian cDNA clone, reported to be PAV-specific (pBY82, Waterhouse, Gerlach & Miller, 1986), hybridised with Swedish MAV-like but not with PAV-like isolates. Probes prepared from the clones detected virus in plant extracts by dot-blot hybridisation with sensitivity greater than that of ELISA. Virus was also readily detected in extracts of viruliferous aphids.     

黑木耳核糖体失活蛋白cDNA 3′-端的克隆与分析

为了克隆黑木耳核糖体失活蛋白cDNA 3′-端,根据随机测得的中间一段蛋白质序列设计简并引物,应用RT-PCR和3′-RACE反应方法,将得到的基因片段与质粒连接,并转化至大肠杆菌中,进行蓝白筛选,再通过琼脂糖凝胶电泳法和PCR法验证白斑,从而得到阳性重组质粒,最后克隆出该目的蛋白基因片段。结果表明,克隆出的cDNA 3′-端为330bp的开放阅读框(ORF),编码107个氨基酸和2个终止密码子。经试验证明和文献检索,克隆得到的cDNA 3′-端为一种新基因。     

Replication and packaging of Turnip yellow mosaic virus RNA containing Flock house virus RNA1 sequence

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. In this study, RNA1 sequence of Flock house virus (FHV) was inserted into the TYMV genome to test whether TYMV can accommodate and express another viral entity. In the resulting construct, designated TY-FHV, the FHV RNA1 sequence was expressed as a TYMV subgenomic RNA. Northern analysis of the Nicotiana benthamiana leaves agroinfiltrated with the TY-FHV showed that both genomic and subgenomic FHV RNAs were abundantly produced. This indicates that the FHV RNA1 sequence was correctly expressed and translated to produce a functional FHV replicase. Although these FHV RNAs were not encapsidated, the FHV RNA having a TYMV CP sequence at the 3’-end was efficiently encapsidated. When an eGFP gene was inserted into the B2 ORF of the FHV sequence, a fusion protein of B2-eGFP was produced as expected. [BMB Reports 2014; 47(6): 330-335]     

The Ustilago maydis virally encoded KP1 killer toxin

Some strains of the plant-pathogenic fungus Ustilago maydis secrete toxins (killer toxins) that are lethal to susceptible strains of the same fungus. There are three well-characterized killer toxins in U. maydis–KP1, KP4, and KP6–which are secreted by the P1, P4, and P6 subtypes, respectively. These killer toxins are small polypeptides encoded by segments of an endogenous, persistent double-stranded RNA (dsRNA) virus in each U. maydis subtype. In P4 and P6, the M2 dsRNA segment encodes the toxin. In this work, the KP1 killer toxin was purified for internal amino acid sequence analysis, and P1M2 was identified as the KP1 toxin-encoding segment by sequence analysis of cDNA clones. The KP1 toxin is a monomer with a predicted molecular weight of 13.4kDa and does not have extensive sequence similarity with other viral anti-fungal toxins. The P1M2 segment is different from the P4 and P6 toxin-encoding dsRNA segments in that the 3’non-coding region of its plus strand has no sequence homology to the 3’ends of the plus strands of P1M1, P4M2, or P6M2.     

椰心叶甲CYP4基因的克隆及绿僵菌侵染后的诱导表达研究

根据细胞色素P450家族4（CYP4）的氨基酸保守序列设计1对简并引物,从椰心叶甲Brontispa longissima成虫总RNA中扩增得到5个cDNA片段（GenBank登录号: DQ238840－DQ238844）。以3′-RACE法获得片段BLWH4的3′端序列,推导的氨基酸序列表明其结构中含有CYP家族的特征性保守序列： 螺旋K区的ETLR和血红素结合区的F××G×××C×G。以18S 为对照的RT-PCR分析表明,BLWH4在成虫的mRNA表达量远大于幼虫。绿僵菌Metarhizium anisopliae菌株MA-3和MA-4侵染椰心叶甲成虫及5龄幼虫后,BLWH4的mRNA表达增强,提示BLWH4可能具有增强椰心叶甲抵抗绿僵菌侵染的作用。     

Potato virus X-related single- and double-stranded RNAs: Characterization and identification of terminal structures

Six species of 3′-coterminal poly(A) -containing RNAs of subgenomic (sg) size have been found in plants infected with potato virus X (PVX): two major (0.9 kb — the coat protein mRNA, and 2.1 kb) and four minor (1.4, 1.8, 3.0 and 3.6 kb). The 5′-end of the shortest sgRNA is located 26 nucleotides upstream of the initiating codon of the coat protein gene (812 nucleotides from the 3′-terminal poly(A) tract of the PVX genomic RNA). Double-stranded analogues have been found for most sgRNAs. The genomic-size double-stranded RNA (the replicative form) is shown to carry a poly(A)-poly(U) hybrid of a predominant length of 150–250 bp on one end, and an unpaired G residue on the other (the 3′-end of the negative chain). In contrast to this(—) the chains of double-stranded 0.9 and 2.1 kbp sgRNAs lack the unpaired G and both end in C.     

Tomato extensin and extensin-like cDNAs: structure and expression in response to wounding

Two tomato cDNA libraries were synthesized from poly(A)⁺ RNAs isolated from unwounded and wounded tomato stems. These cDNA libraries were packaged in gt10 and screened by in situ plaque hybridization with a tomato extensin gene clone (pTom 5.10). Several cDNA clones were identified and isolated from both libraries in this manner and subjected to restriction enzyme digestion, Southern gel blot hybridization, RNA gel blot hybridization, and DNA sequence analyses. From these analyses, the various cDNA clones were found to fall into one of five distinct classes (classes I–V). Class I clones hybridized to a 4.0 kb mRNA which accumulated markedly after wounding and encoded an extensin characterized largely by Ser-(Pro)₄-Ser-Pro-Ser-(Pro)₄-(Tyr)₃-Lys repeats. Class II clones hybridized to a 2.6 kb mRNA which showed no accumulation following wounding and encoded an extensin containing Ser-(Pro)₄-Ser-Pro-Ser-(Pro)₄-Thr-(Tyr)_1–3-Ser repeats. Class III clones hybridized to a 0.6 kb mRNA which greatly accumulated in response to wounding and encoded a glycine-rich protein (GRP) with (Gly)_2–6-Tyr-Pro and(Gly)_2–6-Arg repeats. Class IV clones contained both class I and class III DNA sequences and consequently hybridized to both the 4.0 kb and the 0.6 kb wound-accumulating mRNAs; these clones encoded a portion of a GRP sequence on one DNA strand and encoded a portion of an extensin sequence on the other DNA strand. Class V clones hybridized to a 2.3 kb mRNA which decreased following wounding and encoded a GRP sequence characterized by (Gly)_2–5-Arg repeats.