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Temperature-sensitive mutants of the adenovirus 2-simian virus 40 hybrid, Ad2+ ND1‡, were isolated and crossed with temperature-sensitive mutants of adenovirus type 5 (Ad5). Forty-five wild type recombinants were selected and their DNAs were separately digested with five restriction endonucleases, EcoRI, HpaI, BamI, HindIII and SmaI. There are 15 sites cleaved by these enzymes that are unique to one parental DNA or the other. The fragments obtained by digestion of each of the recombinant DNAs were separated by gel electrophoresis and compared with those obtained from the parental genomes. The recombinant DNAs were found to consist of sequences derived both from Ad5 and from Ad2+ND1. Knowing the positions at which the five restriction enzymes cleave the genomes of the parental serotypes, it was possible to decide which regions of each recombinant DNA are composed of Ad5 and which of Ad2+ND1 sequences. In this way the cross-over events that generated the recombinants were mapped and the physical locations of the ts mutations in the parental genomes were determined.

Restriction endonuclease EcoRII makes about twenty double-stranded breaks per molecule of λh80 DNA. The 5′-terminal sequences are pC-C-A-G-G and pC-C-T-G-G. These are complementary and rotationally symmetrical, showing how the enzyme may produce DNA fragments with short cohesive ends.

Leptospirosis is a bacterial zoonotic disease caused by spirochetes of the genus Leptospira that affects human and a wide range of animals. The direct method of diagnosis of leptospirosis, has been so far by culture isolation but it is time consuming and potentially biohazardous. Another traditional method is the detection of antibodies (Serological tests) which is also a time consuming method and fails to identify the infecting serovar. To overcome these limitations associated with the cultivation and serology, we developed PCR assay targeting partial lipL21 gene and lipL41 gene of Leptospires using in-house designed P28/29 and P30/31 primers, with a product size of 385bp and 427bp. The amplicons were subjected to restriction enzyme digestion using RsaI, Pvu II and HindIII for product of P28/29 and ClaI, TaqI and RsaI were used for product of P30/31. The protocols were standardized and the assay targeting the partial lipL21 and lipL41 gene was found to be specific for eight pathogenic Leptospires out of nine leptospires tested. The products were then cloned in pGEMT Easy vector and sequenced to facilitate further studies. PCR could detect the target bacterial gene without any ambiguity and showed good efficiency in detection of targeted species in the sample. This simple, rapid and cost-effective method can be applicable in a prediction system to prevent disease outbreak by these Leptospira species and can be considered as an effective tool for disease diagnosis of Leptospira species. Key words: PCR; lipL21; lipL41; Molecular Diagnostics; Transformation. Nepal Journal of Biotechnology. Jan. 2011, Vol. 1, No. 1 : 22-30

The restriction endonuclease, Hin dIII, gives rise to three fragments in each of the three mitochondrial DNAs isolated from the established mammalian cell lines LA9 (mouse), HeLa (human), and BSC-1 (African green monkey). The restriction endonuclease, Eco RI, gives rise to three fragments in mitochondrial DNA from HeLa and to two in DNAs from LA9 and BSC-1. The sizes and the orders of the fragments in the respective genomes have been determined with data obtained from the electron microscope. The origin and the direction of replication have been designated in each of the cleavage maps. Polyacrylamide gel electrophoretic analyses demonstrated that additional fragments not detectable in the electron microscope and larger than 50 nucleotide pairs were not present.

Deleted genomes of simian virus 40 have been constructed by enzymatic excision of specific segments of DNA from the genome of wild-type SV40 † . For this purpose, a restriction endonuclease from Hemophilus influenzae (endo R · Hind III) was used. This enzyme cleaves SV40 DNA into six fragments, which have cohesive termini. Partial digest products were separated by electrophoresis in agarose gel and subsequently cloned by plaque formation in the presence of complementing temperature-sensitive mutants of SV40. Individual deletion mutants generated in this way were mapped by analysis of DNA fragments produced by endo R · Hin d digestion of their deleted genomes, and by heteroduplex mapping. Two types of deletions were found: (1) “excisional” deletions, in which the limits of the deleted segment corresponded to Hin dIII cleavage sites, and (2) “extended” deletions, in which the deleted segment extended beyond Hin dIII cleavage sites. Excisionally deleted genomes presumably arose by cyclization of a linear fragment via cohesive termini generated by endo R · Hin dIII whereas genomes with extended deletions probably were generated by intramolecular recombination near the ends of linear fragments. Of the nine mutants analyzed, two had deletions in the “early” region of the SV40 genome, six had deletions in the “late” region, and one had a deletion that spanned both regions.