PCR amplification of large genomic fragments from human and simian immunodeficiency virus infected cell lines.

Polymerase chain reaction (PCR) has been used to amplify the large fragments from viral genomic DNA of SIV from wild caught, asymptomatic Erythrocebus monkeys from Western Africa (Senegal) and also from HIV-2 infected cell lines. By using consensus primer sequences from highly conserved stretches of gag, pol and env genes, two halves of the viral genome of HIV-2 and SIV (isolated from west African Erythrocebus monkeys) have amplified by PCR. One half spans 5200 bp from within the U3 region of the 5' long terminal repeat (LTR) into pol gene and an overlapping fragment spans 3700 bp from the pol gene into U5 region of 3' LTR. Also fragments ranging from 1-2.3 kb from gag pol and env genes have been successfully amplified. Our data demonstrate that primers used to amplify large segments from viral DNA yield better results if they are derived from a consensus sequence of a highly conserved stretch of the viral genome.     

Sequence organization of feline leukemia virus DNA in infected cells

A restriction site map has been deduced of unintegrated and integrated FeLV viral DNA found in human RD cells after experimental infection with the Gardner-Arnstein strain of FeLV. Restriction fragments were ordered by single and double enzyme digests followed by Southern transfer (1) and hybridization with 32P-labeled viral cDNA probes. The restriction map was oriented with respect to the 5' and 3' ends of viral RNA by using a 3' specific hybridization probe. The major form of unintegrated viral DNA found was a 8.7 kb linear DNA molecule bearing a 450 bp direct long terminal redundancy (LTR) derived from both 5' and 3' viral RNA sequences. Minor, circular forms, 8.7 kb and 8.2 kb in length were also detected, the larger one probably containing two adjacent copies of the LTR and the smaller one containing one comtaining one copy of the LTR. Integrated copies of FeLV are colinear with the unintegrated linear form and contain the KpnI and SmaI sites found in each LTR.     

Long terminal repeat of murine retroviral DNAs: sequence analysis, host-proviral junctions, and preintegration site.

The nucleotide sequence of the long terminal repeat (LTR) of three murine retroviral DNAs has been determined. The data indicate that the U5 region (sequences originating from the 5' end of the genome) of various LTRs is more conserved than the U3 region (sequences from the 3' end of the genome). The location and sequence of the control elements such as the 5' cap, "TATA-like" sequences, "CCAAT-box," and presumptive polyadenylic acid addition signal AATAAA in the various LTRs are nearly identical. Some murine retroviral DNAs contain a duplication of sequences within the LTR ranging in size from 58 to 100 base pairs. A variant of molecularly cloned Moloney murine sarcoma virus DNA in which one of the two LTRs integrated into the viral DNA was also analyzed. A 4-base-pair duplication was generated at the site of integration of LTR in the viral DNA. The host-viral junction of two molecularly cloned AKR-murine leukemia virus DNAs (clones 623 and 614) was determined. In the case of AKR-623 DNA, a 3- or 4-base-pair direct repeat of cellular sequences flanking the viral DNA was observed. However, AKR-614 DNA contained a 5-base-pair repeat of cellular sequences. The nucleotide sequence of the preintegration site of AKR-623 DNA revealed that the cellular sequences duplicated during integration are present only once. Finally, a striking homology between the sequences flanking the preintegration site and viral LTRs was observed.     

Molecular cloning of unintegrated visna viral DNA and characterization of frequent deletions in the 3' terminus

Visna viral DNA, like other retroviral DNA, exists in two circular forms in infected cells. The larger probably contains two copies of the LTR, the smaller, one copy. Recombinant DNA techniques were used to clone unintegrated circular visna viral DNA in the lambda WES . lambda B vector. Circular visna viral DNA was digested with the restriction enzyme SstI, which yields a 9.2-kb viral DNA fragment containing 90% of the viral genome colinear with the restriction map of linear viral DNA. This fragment extends from a site about 900 bp from the left (5') end of the viral DNA molecule, through the 3' region, including U3 and R sequences at its right (3') end. The recombinant clones isolated contain visna viral DNA inserts which range in size from 3.1 kb to 9.2 kb. All the clones contain the 5' region intact, but most had sustained deletions of varying lengths in the 3' terminal region of the cloned fragment.     

通过PCR进行长片段DNA的合成

利用PCR合成DNA长片段(Synthesis Large Frament DNA using PCR,SLFD PCR)是一种有效的合成长片段DNA的方法。采用一段已知的500～600bp碱基的DNA片段为PCR模板,根据所要合成的DNA序列可以设计一系列的PCR引物,这些引物都位于模板DNA的5’端,长度为50～60bp,且从5’到3’方向顺序重叠,重叠碱基数目为12～15,全部引物叠加所得到的DNA正是自己所要合成的DNA。这组引物中最3’端的一条含有一个BamH Ⅰ酶切位点,在该位点后面有15碱基与模板DNA5’端一致的序列。另外还设计一条与该模板匹配的下游引物,引物内也含有一个BamH Ⅰ酶切位点。首先采用5’端最右侧的引物与下游引物进行PCR,在PCR进行10个循环后,以此次PCR的产物为下一轮PCR的模板,该轮PCR采用右侧倒数第二个引物为上游引物,下游引物保持不变。采用类似的方法,完成所有的PCR循环,就可以得到所需要合成的DNA长片段。该方法尤其适合100～200碱基左右的长片段DNA的快速合成与克隆。     

Nucleotide sequence of the 3' end of MCF 247 murine leukemia virus

We isolated DNA clones of MCF 247, a leukemogenic, recombinant type C virus obtained from the thymus of an AKR mouse. We determined the nucleotide sequence of the viral long terminal repeat (LTR) and the 3' end of env, and we compared the sequences to corresponding sequences of the genome of Akv virus, the putative ecotropic parent of MCF 247. By analogy with Moloney leukemia virus, we identified the amino terminus of Prp15E, the C-terminal proteolytic cleavage product of env and precursor to mature virion p15E. In MCF 247 the presumptive Prp15E is encoded by a 603-nucleotide open reading frame. The majority of this sequence is identical to that of Akv. However, a recombination event near the 3' end of the Prp15E-coding region introduces nonecotropic sequences into MCF 247, and these extend to the 3' end through the U3 portion of the LTR. The U3 regions of Akv and MCF 247 are about 83% homologous. The R and U5 regions of the LTR of MCF 247 and Akv are identical. Large RNase T1-resistant oligonucleotides analyzed previously in numerous ecotropic and MCF viral genomes were located within the Akv and MCF 247 DNA sequences. The resulting precise T1 oligonucleotide maps of the 3' ends of MCF viral genomes reveal that the biologically defined, leukemogenic class I MCFs isolated from thymic neoplasms of inbred mice all share the sequence pattern seen in MCF 247, a representative of this group; they possess recombinant Prp15E genes and derive U3 from their nonecotropic parents.     

禽痘疫苗病毒中网状内皮组织增生病病毒5''''LTR整合位点序列分析

参照国外发表的禽网状内皮组织增生病病毒（REV）5’长末端重复序列（LTR）在禽痘病毒（FPV）疫苗株基因组上的整合位点及相关序列,合成一对来自FPV的引物,从国内5个不同厂家生产的禽痘疫苗中经PCR均扩增到REV-5’LTR。通过序列比较发现,我国5个FPV疫苗毒株中REV-5’LTR整合位点与美国和澳大利亚的天然重组禽痘疫苗完全一致。其中,有3个的REV-5’LTR插入序列也与美国的Vac-3-Am株和澳大利亚的Vac-M3-Au株有100％的同源性。另2个中国疫苗毒株中的REV．LTR插入序列与美国疫苗毒株Vac-1-A。中的REV-LTR插入序列有99．6％的同源性。但是,这5个中国禽痘疫苗毒株中整合的REVLTR与中国近年分离到的REV野毒株HA9901的5’LTR的同源性只有75．4％-92．4％。     

Efficient replication, integration, and packaging of retroviral vectors with modified long terminal repeats containing the packaging signal.

Retroviral vectors were modified to contain packaging (psi) signals of varying lengths (nucleotides 211-355, 211-565, or 211-1039 of MoMuLV RNA) between the U3-r and U5 sequences of their 5' long terminal repeat (LTR). For the vector MoTN-PR3, containing the full length 211-1039 nucleotide-long psi signal within the 5' LTR, replication, integration, and packaging were almost as efficient as for the original unmodified vector. This result confirmed that the 211-1039 nucleotide-long sequence from the MoMuLV RNA is sufficient and necessary to allow efficient packaging of RNAs. In addition, an important site was revealed where insertion of foreign DNA sequences of up to 829 nucleotides can be made within the 5' LTR, between U3-r and U5 sequences, without affecting viral replication, integration, or packaging.     

Retroviral DNA integration: structure of an integration intermediate

The structure of a presumptive DNA intermediate in the integration of retroviral DNA was studied in a cell-free reaction with exogenously added target DNA. The product made by viral core particles of Moloney murine leukemia virus (Mo-MLV) containing linear viral DNA has a structure consistent with an integration mechanism similar to that observed during bacteriophage Mu transposition. In this intermediate, the 3' ends of the LTR sequences are joined to the target DNA, while the 5' ends of the viral DNA remain unjoined. The 5' ends of the LTR sequences in the intermediate are exactly the same as those found in the unintegrated linear double-stranded viral DNA. This result demonstrates that the linear form of Mo-MLV DNA can integrate directly without prior circularization.     

猪PAC克隆的微卫星DNA分离研究

利用(CA)8核心序列,设计锚定引物,采用直接测序法,从单个猪PAC克隆中分离到一个新微卫星DNA。根据该微卫星DNA的侧翼序列,设计了专一引物,在8个猪种40个个体中检测到3个等位基因,片段长度分别为305 bp、307 bp和309 bp。3种等位基因纯合子个体的PCR产物序列分析表明,这3种等位基因分别有12、13和14次CA双核苷酸重复。 Abstract:A novel microsatellite DNA was isolated from a single porcine PAC clone by sequencing the PAC clone directly with (CA)n repeat motif anchored primer.The specific primer pairs flanking the (CA)n repeat region were used to amplify the genomic DNA of 40 individuals from 8 pig breeds,which detected three alleles with the fragment length of 305 bp,307 bp and 309 bp.The PCR product sequencing results of homozygous animals representing three alleles revealed that those three alleles contained 12,13 and 14 CA dinucleotide repeats respectively.     

应用RACE法克隆鸽恒定链基因的研究

为比较禽类恒定链的结构和功能, 应用RACE (Rapid Amplification of cDNA Ends) 技术首次克隆并鉴定了鸽恒定链基因。首先用一对含高度保守的DNA片段的简并引物, 从鸽脾细胞RNA扩增部分恒定链片段, 接着测序并设计新引物分别从5′和3′RACE扩增延长该片段。最后根据全基因的序列设计上、下游引物,获得大小为1 050 bp的全长cDNA。比较核苷酸序列, 鸽与鸡的Ii链同源性达到82.8％, 而与人等其它动物的同源性则在52.0％以上; 其中633 bp的开放阅读框编码211个氨基酸残基的前体蛋白。推导和分析氨基酸序列表明, 分子结构与鸡恒定链相似, 其中有些氨基酸残基表现出较高的保守性。     

Investigation of diversity and isotypes of the beta-tubulin cDNA in several small strongyle (Cyathostominae) species

The diversity of the beta-tubulin cDNAs of the cyathostominae and the occurrence of further isotypes were examined in adult worms isolated from an anthelmintic-na?ve horse. cDNAs encoding beta-tubulin from Cyathostomum catinatum, Cylicocyclus nassatus, Cylicocyclus insigne, Cylicocyclus radiatus, Cylicocyclus elongatus, Cyathostomum coronatum, and Cyathostomum pateratum were characterized using specific primers developed from the cDNA sequence of Cc. nassatus. The cDNA sequences span 1,429 bp and show identities ranging from 95.6 to 100%. The deduced protein sequences span 448 amino acids and were 98-100% identical. The amino acid sequences of the 7 species varied within and between species at 10 positions. A 3' Rapid Amplification of cDNA ends using a degenerate forward primer was carried out with cDNA from Cy. pateratum, Cy. coronatum, Cy. catinatum, and Cc. nassatus to investigate the occurrence of further beta-tubulin isotypes. The expected polymerase chain reaction (PCR) product of 400 bp, including 306 bp of coding sequence, was amplified, as was an additional fragment of 600 nucleotides in the case of Cy. pateratum, Cy. coronatum, and Cy. catinatum. Sequencing of the PCR products revealed no evidence for the existence of a second beta-tubulin isotype in cyathostomes. The variation in size was caused by a length polymorphism within the 3' untranslated region, and 2 functional mRNAs seem to be transcribed from the same gene.     

The R-U5-5' leader sequence of neurovirulent wild mouse retrovirus contains an element controlling the incubation period of neurodegenerative disease.

The wild mouse ecotropic retrovirus CasBrE causes a spongiform neurodegenerative disease after neonatal inoculation, with an incubation period ranging from 2 to 12 months. We previously showed that introduction of long terminal repeat (LTR) and gag-pol sequences from a strain of Friend murine leukemia virus (FB29) resulted in a dramatic acceleration of the onset of the disease. The chimeric virus FrCasE, which consisted of the FB29 genome containing 3' pol and env sequences from the wild mouse virus, induced a highly predictable, lethal neurodegenerative disease with an incubation period of only 16 days. Here we report that the sequences which are primary determinants of the length of the incubation period are located in the 5' end of the viral genome between a KpnI site in the R region of the LTR and a PstI site immediately 5' of the start codon for pr65gag (R-U5-5' leader). This region contains the tRNA primer binding site, splice donor site for the subgenomic env mRNA, and the packaging sequence. Computer-assisted sequence analysis failed to find evidence of a consensus sequence for a DNA enhancer in this region. In addition, sequences within a region of the genome between a ClaI site at the 3' end of env to the KpnI site in the R region of the LTR (inclusive of U3) also influenced the incubation period of the disease, but the effect was distinctly weaker than that of the R-U5-5' leader sequence. This U3 effect, however, appeared to be independent of the number of direct repeats, since deletion of one of two duplicated 42-base repeats containing consensus sequences of nuclear-factor binding domains had no effect on the incubation period of the disease. On the basis of Southern blot analysis of total viral DNA in the tissues, the effect of these sequences on the incubation period appeared to be related to the level of virus replication in the central nervous system. All of the chimeric viruses analyzed, irrespective of neurovirulence, replicated to comparable levels in the spleen and induced comparable levels of viremia.     

PCR detection of Ti and Ri plasmids from phytopathogenic Agrobacterium strains.

A universal primer set (VCF/VCR) for PCR analysis based on the sequences of the virC operon located on Ti and Ri plasmids was designed to detect these plasmids from phytopathogenic Agrobacterium strains. With the VCF (sequence, 5'-ATCATTTGTAGCGACT-3') and VCR (sequence, 5'-AGCTCAAACCTGCTTC-3') primer set, DNA fragments of 730 bp in length were amplified from cell lysates of 10 rhizogenic and 65 tumorigenic agrobacteria. DNA sequencing and Southern hybridization analysis confirmed that the amplified fragments corresponded to the target region. The PCR method is considered convenient for routine determination of the potential pathogenicity of Agrobacterium strains.