Showing papers on "XhoI published in 1994"

Sequence variation in the Epstein-Barr virus latent membrane protein 1.

Abstract: The sequence of the latent membrane protein 1 (LMP-1) gene was analysed in Epstein—Barr virus (EBV) isolates from specific regions representing both type 1 and type 2 EBV. A predominant strain marked by an XhoI restriction enzyme polymorphism (REP) within the LMP-1 gene has been identified in type 1 EBV in nasopharyngeal carcinoma (NPC) from Southern China. This polymorphism was also present in type 2 EBV in NPC from Alaska. In this study, the sequence of the LMP-1 gene was determined in these samples representing type 1 and type 2 EBV and was compared with the prototype lymphoid strains. Consistent nucleotide variation in the amino terminus of LMP-1 was identified in strains marked by the XhoI REP. These changes were present in both EBV type 1 and type 2 strains. Three types of sequence variation were detected in the carboxy terminus of LMP-1. The LMP-1 sequences differed in the number of an 11 amino acid repeat element. In the prototype EBV type 1 (B95-8) sequence and in the type 1 Raji and type 2 HR-1 strains, the third repeat element contained an insertion of 5 amino acids that were also the first five unique amino acids after the last partial repeat element. The third variation was a deletion of amino acids 343 to 352 of the B95-8 LMP-1. This deletion was detected in the type 1 Chinese EBV strains, but was not detected in the type 2 Alaskan strains although the Chinese and Alaskan strains have nearly identical amino acid changes at the amino terminus. Numerous other amino acid changes were detected in the carboxy terminus which did not cosegregate with either EBV type, amino acid changes in the amino terminus, or specific geographic regions. These data indicate that EBV strains can be distinguished by sequence differences within LMP-1 and that unlike the divergence between type 1 and type 2 EBV in Epstein—Barr nuclear antigen sequences, different EBV types are nearly identical in LMP-1 sequence.

PCR with end trimming and cassette ligation: a rapid method to clone exon-intron boundaries and a 5'-upstream sequence of genomic DNA based on a cDNA sequence.

Abstract: We described a method for PCR amplification of unknown flanking genomic DNA fragments. This method is a combination of PCR with "end-trimming method" and "cassettes and cassette-primers method". In this method, genomic DNA was digested with three different groups of restriction enzymes. DNA in group 1 was digested with BamHI, BglII, FbaI, or MboI. DNA in group 2 was digested with BlnI, NheI, SpeI, or XbaI. DNA in group 3 was digested with SalI or XhoI. Digested DNA in each group was end-trimmed with Klenow fragment of DNA polymerase I in the presence of only one dNTP; dGTP, dCTP, and dTTP for group 1, 2, and 3, respectively. The synthesized cassettes, C1, C2, and C3, had 5'protruding sequences of 5'-ATC-3',5'-TAG-3', and 5'-CGA-3', respectively. Each compatible cassette was ligated to the end-trimmed DNAs in group 1-3, respectively. Nested PCR was then performed using an end-trimmed and cassette-ligated DNA as a template. Primers annealing to known sequences and cassettes were used for the nested PCR. The amplified DNA fragments were electrophoresed on a polyacrylamide gel and purified. The sequences of the DNA fragments were determined after cloning into pBluescript.

Restriction map of the genomic DNA of Lactobacillus casei bacteriophage PL-1 and nucleotide sequence of its cohesive single-stranded ends

Abstract: A restriction map of the genomic DNA of Lactobacillus casei phage PL-1 was constructed using the restriction endonucleases BamHI, EcoRI, HindIII, KpnI, NruI and XhoI. The PL-1 genome was 42.2 kb in size and had complementary cohesive ends forming a ring-like monomer. The cohesive ends, analysed with exonuclease III and S1 nuclease, were 3′-terminated single strands protruding from both ends. The nucleotide sequence of the cohesive ends, determined by the dideoxynucleotide method, comprised four A + T and 10 G + C pairs: 5′ GAGGCCGACCGTTC 3′/3′ CTCCGGCTGGCAAG 5′. Thus, the cohesive ends of PL-1 DNA were higher in G + C content than those of other known bacteriophage DNAs.

Chloroplast DNA differences between cultivated hop, Humulus lupulus and the related species H. japonicus.

Abstract: Chloroplast DNA (cpDNA) of Humulus Lupulus and H. japonicus was examined by restriction endonuclease analysis with BamHI, BanI, BclI, BstEII, DraI, EcoRI, EcoRV, HindIII, KpnI, PaeR7I, PstI, PvuII, SalI and XhoI. The restriction fragment patterns showed that the cpDNAs shared a large number of restriction sites. However, the chloroplast genomes of the two species could be distinguished by differences in restriction site and restriction fragment patterns in the PstI, PvuII, BclI, EcoRV, DraI and HindIII digests. On the basis of the complexity of restriction enzyme patterns, the enzymes PstI, PvuII, SalI, KpnI and XhoI were selected for mapping the chloroplast genomes. Single and double restriction enzyme digests of cpDNA from the two species were hybridized to cpDNA probes of barley and tobacco. The data obtained from molecular hybridization experiments were used to construct the cleavage site maps. Except for the PstI digest, the arrangement of cpDNA restriction sites was found to be the same for both species. An extra PstI site was present in H. lupulus. Three small insertions/deletions of about 0.8 kbp each were detected in the chloroplast genomes of the two species. Two of these insertions/deletions were present in the large and one in the small singlecopy region of the chloroplast genome. The cpDNA of Humulus was found to be a circular molecule of approximately 148 kbp that contains two inverted repeat regions of 23 kbp each, a small and a large single -copy region of approximately 20 kbp and 81 kbp, respectively. The chloroplast genome of hop has the same physical and structural organization as that found in most angiosperms.

Sizing the Fusobacterium nucleatum genome by pulsed-field gel electrophoresis

Abstract: The genome sizes of Fusobacterium nucleatum strains F1, F3, F6, ATCC 10953, ATCC 25586 and Fev1 were determined by pulsed-field gel electrophoresis (PFGE). The restriction enzymes SmaI, SacII, SalI and XhoI were found to generate a reasonable number of DNA fragments which could be separated by PFGE in agarose gels. The apparent chromosomal lengths of the F. nucleatum strains were determined to be approximately 2.4 million base pairs. This was within the size-range found by experiments exploiting renaturation kinetics.

紀伊水道海域のメイタガレイ2型(ホンメイタとバケメイタ)のmtDNA切断型における遺伝的分化

Abstract: Genetic differentiation between two forms of flatfish, differentially called Honmeita and Bakemeita found in the Kiisuido Channel waters off the Pacific Coast of Central Japan, was investigated by restriction fragment analysis of mitochondrial DNA (mtDNA). 36 specimens of Honmeita and 32 specimens of Bakemeita were analyzed. We demonstrated 17 restriction fragment types using five 6-base recognized restriction endonucleases (BamHI, BglII, HindIII, PstI, XhoI). Five haplotypes in Honmeita and 3 types in Bakemeita were recognized, and there were no common types between the two forms. Nucleotide divergences among haplotypes of flatfishes were estimated to be 0.0074-0.0159, 0.0055-0.0115, 0.0684-0.0948, in the Honmeita form, in the Bakemeita form and between the two forms respectively. Interformal mtDNA nucleotide divergence was estimated to be 0.081. This value suggests that these two forms differentiate so highly from each other at the interspecific level. Intraformal mtDNA nucleotide divergence was estimated to be 0.0016 and 0.0012 in the Honmeita and Bakemeita forms respectively.

A kinetic method of determining the frequency of homologous recombination of plasmids in Escherichia coli cells

Abstract: To test the frequency of recombination by the RecF pathway the hybrid plasmids have been constructed which allow the bioluminescence recombination assay in the transformed E. coli cells. pF2(+) and pF6(+) are derivatives of pUC18 and pACYC184 respectively, with the luxA and luxB genes of Vibrio fischeri cloned downstream of the lac promoter. The luxA genes in pF2(+) and pF6(+) were mutated at the XhoI site and the HindIII site, accordingly, pF8 is a pACYC184 derivative with two copies luxA and luxB genes. The one copy of luxA gene was mutated at the XhoI site and another copy of the luxA gene was mutated at the HindIII site. A kinetic analysis of the population of the replicating and recombinating plasmids has been carried out. Experimental values of the frequency of recombination per one generation (P) were determined for the different E. coli strains.