The complete genomic sequence of egg drop syndrome virus strain AAV-2

In the search for the genome of egg drop syndrome virus (EDSV-76) Chinese strain AAV-2, part of restrietion endonuclease physical map is analyzed, the complete genomic library is organized. On basis of this, the eomplete genome nueleotide sequences (32 838 bp in length, including terminal structures) are determined. The data analysis shows: compared with the other Adenoviruses, strain AAV-2 has more disparity on ganomic structure and the distribution of open reading frame (ORF). There are no elear E1, E3 and F4 regions in AAV-2 genome. Two segments located at both ends of genome (1.1 kb and 8.3 kb in length respectively) have no homology with the other adenovirus genomes. In addition, strain AAV-2 genome lacks ORFs encoding E1A, pⅤ and pⅨ, which are common ORFs encoding early, lately proteins in Adenovirus. This reveals differences between EDSA-76, the sole standard strain of group Ⅲ Avian Adenoviruses, and the other Avian Adenoviruses for the first time. It will help the search for Avian Adenovirus     

A family of moderately repetitive sequences in mouse DNA.

When mouse DNA is digested to completion with restriction endonuclease Eco R1, a distinct band of 1.3 kb segments comprising about 0.5-3% of the genome is observed upon agarose gel electrophoresis. This DNA is not tandemly repeated in the genome and is not derived from mouse satellite DNA. Restriction endonuclease analysis suggested that the 1.3 kb segments are heterogeneous. Specific sequences were selected from the 1.3 kb segments and amplified by cloning in plasmid pBR322. Southern transfer experiments indicated that three separately cloned mouse DNA inserts hybridized predominantly to the Eco R1 1.3 kb band and to the conspicuous subsegments generated by secondary restriction endonuclease cleavage of the sucrose gradient purified 1.3 kb segments. Segments were also excised by Hha I (Hha I segments) from the chimeric plasmids containing mouse DNA inserts and subjected to restriction endonuclease and cross-hybridization analysis. It was found that the three Hha I segments were different, although two of them exhibited partial sequence homology. Cot analysis indicated that each of the Hha I segments are repeated about 10(4) times in the mouse genome. These findings indicate that a family of related but non-identical, moderately repetitive DNA sequences, rather than a single homogeneous repeat, is present in the 1.3 kb Eco R1 band.     

长春和北京地区引起婴幼儿肺炎的3型与7型腺病毒的分子流行病学研究

对长春和北京地区连续12年(1976年冬至1988年春)引起小儿肺炎的3、7型腺病毒102株标本,进行了限制性内切酶核酸电泳图谱分析。56株7型腺病毒经BamHⅠ、BclⅠ、BglⅠ、XbaⅠ、SmaⅠ、HindⅢ分析后,表现为两个基因组型——Ad7 b和Ad7 d。46株3型腺病毒被Bg1 Ⅱ、BamHⅠ酶解后,表现为 3个基因组型——Ad 3Ⅰ、Ad 3Ⅱ、Ad 3Ⅲ。各基因组型的分布情况是:56株7型腺病毒中,43株为Ad 7 b(76.8％),流行于1976年冬至1986年春;13株是Ad 7 d(23.2％),出现于1982年,与Ad 7 b共同流行;1986年～1988年分析的5株病毒都是Ad 7d。43株3型腺病毒中,Ad3Ⅰ42株(91.0％),分布于12年中;Ad 3Ⅱ、Ad 3Ⅲ各2株,散在分布。此结果表明,国内这12年中引起小儿肺炎的3型腺病毒至少有3个基因组型,7型腺病毒至少有两个基因组型。Ad3Ⅰ和Ad7 b是流行优势基因组型。但自80年代初开始出现Ad7 d以来,有逐年增多的趋势,最近两年的标本又都是Ad7 d,很可能它将取代Ad7 b而成为流行的优势基因组型.     

A NheI macrorestriction map of the Neisseria meningitidis B1940 genome

Abstract A macrorestriction map of the Neisseria meningitidis strain B1940 genome was constructed by two-dimensional pulsed-field gel electrophoresis (2D-PFGE) techniques. Digestion of the genomic DNA with the restriction endonuclease NHe I revealed 15 fragments between 10 kb and 450 kb. The sum of the fragments and resolution of the linearized chromosome yielded a total genome size of about 2.3 Mbp. By overlapping methylation with the Alu I-methylase six Nhe I recognition sites could be blocked. Fragments were ordered by partial/complete 2D-PFGE of genomic DNA with and without prior Alu I methylation, respectively. All nine Alu I-methylase/ Nhe I and 14 Nhe I restriction sites could be mapped on a single circular chromosome. This map will serve as a useful tool for further genetic analysis of meningococci and exemplifies the power of non-radioactive 2D-PFGE techniques to construct large physical genome maps with a single restriction enzyme.     

The nucleotide sequence of the transforming early region E1 of adenovirus type 5 DNA

The sequence of the leftmost 11.3% of the non-oncogenic human adenovirus type 5 (Ad5) DNA has been determined. This segment contains the entire early region E1 of the Ad5 genome which has been shown to be involved in in vitro transformation of non-permissive rodent cells (Van der Eb et al., 1980). From the DNA sequence, and from the mRNA sequence data obtained by Perricaudet et al, (1979, 1980) for the E1 mRNAs from the closely related adenovirus type 2 (Ad2), it is possible to predict the primary structure of the polypeptides encoded by this region. The function of these proteins in cell transformation is discussed. From the positions of mapped restriction endonuclease sites and termini of RNA segments in the nucleotide sequence the length of the Ad5 DNA is estimated to be 36.6 kb.     

Genome analysis of Pseudomonas aeruginosa by field inversion gel electrophoresis

Abstract The genome of Pseudomonas aeruginosa was analysed by digestion with rare-cutting restriction endonucleases and subsequent field inversion gel electrophoresis (FIGE). P. aeruginosa strain PAO and the 17 IATS strains were investigated. Each strain displayed a unique pattern of restriction fragments. Digestion with Dra I and Ssp I yielded, respectively 7–11 and 2–5 fragments of more than 130 kb in size, indicating the non-random occurrence of AT-rich sequences in the P. aeruginosa genome. The genome size of P. aeruginosa PAO was estimated to be (2.2 ± 0.3) × 10⁶ bp. The applications of DNA fingerprinting for gene cloning, construction of a physical chromosome map, and epidemiological studies, are discussed.     

The complete genomic sequence of egg drop syndrome virus strain AAV-2

In the search for the genome of egg drop syndrome virus (EDSV-76) Chinese strain AAV-2, part of restriction endonuclease physical map is analyzed, the complete genomic library is organized. On basis of this, the complete genome nucleotide sequences (32 838 bp in length, including terminal structures) are determined. The data analysis shows: compared with the other Adenoviruses, strain AAV-2 has more disparity on genomic structure and the distribution of open reading frame (ORF). There are no clear E1, E3 and E4 regions in AAV-2 genome. Two segments located at both ends of genome (1.1 kb and 8.3 kb in length respectively) have no homology with the other adenovirus genomes. In addition, strain AAV-2 genome lacks ORFs encoding ElA, pV and pIX, which are common ORFs encoding early, lately proteins in Adenovirus. This reveals differences between EDSA-76, the sole standard strain of group III Avian Adenoviruses, and the other Avian Adenoviruses for the first time. It will help the search for Avian Adenovirus and will also help the search of all Adenoviruses.     

The photobinding of 5,7-dimethoxycoumarin to adenovirus type-2 DNA. A method for in vitro mutagenesis

The photobinding of 5,7-dimethoxycoumarin to isolated adenovirus-type 2 DNA has been investigated with respect to the influence of the ionic environment, and varying molar ratios of DNA_(p): 5,7-dimethoxycoumarin. In particular, the ultraviolet radiation-induced covalent addition of 5,7-dimethoxycoumarin to adenovirus DNA was increased by reducing the concentration of Na⁺. The maximum photobinding of 5,7-[³H]dimethoxycoumarin to adenovirus DNA under the given ionic condition was one 5,7-dimethoxycoumarin per 101 nucleotides. Moreover, restriction enzyme analysis of the 5,7-dimethoxycoumarin-DNA photoadduct versus unmodified viral DNA, suggested that the sequence d(A-T) is the preferential site for intercalation and subsequent photobinding of 5,7-dimethoxycoumarin. This susceptibility of d(A-T) sequences to 5,7-dimethoxycoumarin interaction has a corresponding influence on the survival of adenovirus because of the A-T-rich sequences that occur in some of the early gene regions of the adenovirus genome. Specifically, 5,7-dimethoxycoumarin per 800 nucleotides in adenovirus DNA reduced the surviving fraction of adenovirus to a value of 0.1 after DNA infectivity (transfection) into human 293 cells. Results suggest that 5,7-dimethoxycoumarin may be used for generating a limited ‘library’ of mutations in each of the five early gene regions of the adenovirus genome.     

Identification of mouse adenovirus type 1 early region 1: DNA sequence and a conserved transactivating function.

The left end of the genome of mouse adenovirus type 1 (also known as strain FL) was characterized by determination of the DNA sequence, amino acid similarities with early region proteins of primate adenoviruses, and a functional assay. Several specific DNA sequence features were similar to those found in human adenoviruses, and open reading frames from this region could encode proteins similar to human adenovirus early region 1A and early region 1B proteins. DNAs from this region were tested in transient-expression assays in human and mouse cells were found to transactivate the human adenovirus type 5 early region 3 promoter fused to the chloramphenicol acetyltransferase gene. The data indicate structural and functional homologies between mouse adenovirus type 1 early region 1 and early region 1 of primate adenoviruses.     

Restriction fragment fingerprint and genome sizes of Staphylococcus species using pulsed-field gel electrophoresis and infrequent cleaving enzymes.

Large restriction fragments of genomic DNA from Staphylococcus species were separated by pulsed-field gel electrophoresis (PFGE). Five different strains of S. aureus (ISP8, SAU3A, PS96, ATCC 6538, ATCC 15564) and three representative strains of S. haemolyticus SM102, S. warneri MCS4, S. cohnii LK478 from human hosts, and one strain of S. aureus (ATCC 8432) from an avian host were used in this study. Since Staphylococcus is A + T rich (approximately 67%), restriction fragments were obtained by digesting chromosomal DNA with endonucleases that recognize GC-rich sequences. Five enzymes Csp I, Sma I, Ecl XI, Ksp I, or Sac II were used for generation of few (7 to 16) distinctly separated fragments, with average sizes in the range of 200-300 kb. The size distribution of restriction fragments for each enzyme for each strain produced a strain-identifying fingerprint, and the genome size of each strain was determined from such restriction fragments separated by PFGE.     

Molecular analysis of the mitochondrial genome of Helianthus annuus in relation to cytoplasmic male sterility and phylogeny

Summary A circular supercoiled mitochondrial DNA plasmid P₁ (1.45 kb) is shown in both normal fertile plants of Helianthus annuus, and some cytoplasmic male sterile lines (CMS A and CMS P). In contrast, no plasmid is found in some other types of CMS C, I, B and K. A circular supercoiled DNA (P₂) of higher molecular weight (1.8 kb) is observed in CMS F. The mitochondrial plasmid P₁ was cloned, nick-translated and hybridized with native mitochondrial DNA from different lines of male fertile, CMS or wild Helianthus. No sequence homology has been detected between plasmid DNA P₁ and high molecular weight mitochondrial DNA in any line examined. A slight hybridization occurs between plasmids P₁ and P₂. Thus, there is no apparent relationship between mitochondrial plasmid DNA and CMS or Helianthus species. On the contrary, each Helianthus CMS and male fertile strain can be characterized by digestion fragment patterns (Sal I and Bgl I). Analysis of mitochondrial DNA from wild Helianthus strains indicated a relation between some CMS and the strain from which they were maternally derived, as for example CMS I and H. annuus ssp lenticularis and CMS F and H. petiolaris fallax. On the basis of restriction endonuclease patterns, a CMS phylogenic tree is proposed which illustrates a molecular polymorphism in the mitochondrial genome of Helianthus.     

猪Ⅱ型圆环病毒豫A株的全基因组克隆与序列分析

参照国外发表的猪Ⅱ型圆环病毒(porcine circovirus type 2,PCV-2)全基因组序列,设计一对PCV-2特异性引物,用该室分离的PCV-2豫A株感染PK-15细胞,从中提取PCV-2复制型基因组DNA,并以之为模板进行PCR扩增.回收PCR产物,构建重组测序质粒T-PCV-2.测序结果表明,猪Ⅱ型圆环病毒豫A株的全基因组为1767bp,与GenBank收录的PCV-2国外分离株核苷酸的同源性可高达97%.序列分析表明,复制型豫A株的基因组包含10个读码框架,其中ORF1、ORF2是其两个最主要的读码框架,分别编码314、234个氨基酸.豫A株和PCV-1间的ORF1、ORF2的氨基酸序列同源性分别为85%、66%,与其它PCV-2毒株间的ORF1氨基酸同源性均在98%以上,而ORF2的氨基酸同源性为92%～97%.     

Genetic analysis of adenovirus type 2. VIII. Physical locations of temperature-sensitive mutations.

Temperature-sensitive mutations of human adenoviruses can be physically located on the viral genome by determining the DNA structures of recombinants formed in genetic crosses between different members of the same subgroup. We have analyzed the DNA structures of many interserotypic recombinants formed in crosses between temperature-sensitive (ts) mutants of adenovirus type 2 and adenovirus type 5 with the restriction endonucleases BamHI, EcoRI, HindIII, and and Sma I. In this way, we have mapped the physical coordinates of adenovirus type 2 (Ad2) ts1, Ad2 ts3, Ad2 ts4, and Ad2 ts48, and refined the mapping of Ad5 ts1.     

Chromosome map of the phototrophic anoxygenic bacterium Chromatium vinosum

Abstract The genome of Chromatium vinosum has been characterized using pulsed field gel electrophoresis. Two restriction endonucleases, Ase I and Spe I, generated DNA fragments of size distributions suitable for mapping the genome of the anoxyphotobacterium C. vinosum DSM 180. Ase I produced 24 fragments ranging from 367 to 10.8 kb and Spe I yielded 13 fragments from 720 to 12 kb. A total genome size of 3.674 Mb was determined by summing the fragment lengths in each of the digests generated using the different restriction endonucleases. Intact total DNA from C. vinosum shows the presence of three extrachromosomal elements. Three rRNA regions were located in the strain DSM 180. Restriction patterns of the strain DSM 180 have been compared with ATCC 17899 and DSM 185 of the same species.     

快速获取55型腺病毒基因组序列的方法

【目的】建立快速获取55型腺病毒的全长基因组序列的方法。【方法】根据55型腺病毒的基因组特点,设计覆盖55型腺病毒基因组序列的12对引物,分别以55型腺病毒DNA为模板,扩增得到12个PCR产物,通过对12个PCR产物测序及序列拼接,获得55型腺病毒的全长基因组序列。【结果】从本院急性上呼吸道感染者咽拭子标本中分离得到一株55型腺病毒毒株SF04/SC/2016,以其DNA为模板扩增成功获得12个PCR产物,对其进行测序,并对12段序列进行拼接得到55型腺病毒的全长基因组序列,与已报到的各型腺病毒序列进行比对,采用邻位相连法构建系统发育进化树,所得序列与55型腺病毒处于同一分支,进一步确认该病原体为55型腺病毒。【结论】研究公布的序列和方法,能够实现更方便对腺病毒的快速测序,为揭示55型腺病毒的进化特点及制订疾病防控策略提供了有效手段。     

Molecular cloning of DNA complementary to a mouse α-fetoprotein mRNA sequence

DNA complementary to mouse yolk sac messenger RNA has been inserted at the PstI site of the plasmid pBR322 by annealing of the oligo(dG)-tailed plasmid DNA with the oligo(dC)-tailed mouse DNA. Transformation of Escherichia coli strain RRI with this annealed DNA yielded clones bearing recombinant plasmids. The clones were screened for DNA complementary to mouse a-fetoprotein (AFP) messenger RNA sequences by hybridization with a cDNA probe transcribed from an AFP mRNA of over 90% purity. Out of nine plasmids that were isolated and analyzed by restriction mapping, all had homologous insert DNA of various lengths. The plasmid with the longest insert, pAF6, contained 1.65 kb of added DNA, which is about 70% of the AFP mRNA. This clone was positively identified by a hybridization-translation procedure to contain a cDNA sequence for AFP. A restriction map of this clone and the orientation of the message are presented.     

Identification of a new strain of adenovirus type 2 by restriction endonuclease analysis

Restriction endonuclease analysis has been used to identify a new strain Ad2a of human adenovirus type 2 (Ad2). The sites for six restriction endonucleases have been mapped on the Ad2a genome and compared with those for the Ad2 prototype.     

A novel phage genome integrated into a plasmid in Bacillus thuringiensis strain AF101

Bacillus thuringiensis strain AF101 possesses a single plasmid (pAF101) with a molecular size of 42 MDa (69 kb). During plasmid curing experiments in strain AF101, we found that a phage (J7W-1) was induced by ethidium bromide treatment. Moreover, the phage genome (48 kb) hybridized only with pAF101 on a Southern blot of the DNA of a cleared lysate prepared from strain AF101. Comparison of the restriction patterns of pAF101 and J7W-1 phage DNA revealed that pAF101 contains not only the entire phage DNA but also a plasmid-specific DNA region. These results indicate that the J7W-1 genome has been stably integrated into pAF101 in strain AF101. Integration of the J7W-1 genome into a plasmid was also observed after phage infection of the type strain of B. thuringiensis subsp. israelensis.