From examination of published DNA sequences of genes found inserted at a specific site in integrons, all genes are shown to be associated, at their 3' ends, with a short imperfect inverted repeat sequence, a 59-base element or relative of this element. The similarity of the arrangement of gene inserts in the integron and in the Tn7 transposon family is described. A refined consensus for the 59-base element is reported. Members of this family are highly diverged and the relationship of a group of longer elements to the 59-base elements is demonstrated. The ability of 59-base elements of different length and sequence to act as sites for recombination catalysed by the integron-encoded DNA integrase is demonstrated, confirming that elements of this family have a common function. The ability of elements located between gene pairs to act as recombination sites has also been demonstrated. The recombination cross-over point has been localized to the GTT triplet which is conserved in the core sites, GTTRRRY, found at the 3' end of 59-base elements. Recombination at the core site found in inverse orientation at the 5' end of the 59-base elements was not detected, and the sequences responsible for orientation of the recombination event appear to reside within the 59-base element. A model for site-specific insertion of genes into integrons and Tn7-like transposons is proposed. Circular units consisting of a gene associated with a 59-base element are inserted into an ancestral element which contains neither a gene nor a 59-base element.(ABSTRACT TRUNCATED AT 250 WORDS)

The positions of the outer boundaries of the 5'-and 3'-conserved segment sequences of integrons found at several different locations have been determined. The position of the 5' end of the 5'-conserved segment is the same for six independently located integrons, Inl (R46), In2 (Tn2l), In3 (R388), In4 (Tn1696), In5 (pSCH884), and InO (pVS1). However, the extent of the 3'-conserved segment differs in each integron. Many antibiotic resistance genes found on plasmids and transposons in gram-negative bacteria are located at a unique site within a conserved DNA sequence (7,13,14,31,34). The elements which contain the antibiotic resistance genes are found in several distinct locations (14, 31), and as they are formally distinct from other genetic elements in that they determine site-specific integration functions, a DNA integrase and a recombination site, and are thus able to acquire genes at the specific site, they were named integrons (31). As a consequence of differences in the number, nature, and order of the inserted genes, integrons exist in a large variety of forms (see reference 13). Individual genes are inserted as cassettes (8)(9)(10)13), and each cassette includes a gene and a recombination site belonging to a family of sites known as 59-base elements. Integrons also act as natural expression vectors for the inserted genes, by supplying a promoter located in the conserved sequences upstream of the genes (7,14,31). Two distinct classes of integrons have been identified (13,31). The first class consists of those integrons which are associated with the sulfonamide resistance determinant sulI in a conserved region 3' to the inserted gene cassettes, and most of the cassetteassociated resistance genes that have been isolated from clinical strains are found in this context. The second class is found in Tn7 and related transposons (see reference 13). The two integron classes can be distinguished by the sequences of the DNA integrases that they encode, which share 40% identical amino acids (14). Despite the differences in the integrase proteins, the two classes of integrons can include identical gene cassettes and exchange of cassettes is likely to occur (see reference 13).

The completed sequence and genome organization of OAV287, a serologically distinct ovine adenovirus, is described. The genome of 29,544 bp has inverted terminal repeats that are only 46 bp in length. Many OAV genes are identified by their homology with other adenovirus (Ad) sequences but three groups of reading frames show little homology. One group at the left-hand end of the genome probably represents the E1A/E1B regions. Two others, on the complementary strand at the right-hand end of the genome, are tentatively proposed as the E4 and E3 regions. They are separated by approximately 1 kb of A/T-rich sequence of unknown function with E3 being adjacent to the terminus. Structural proteins V and IX of human Ads are absent from the OAV genome but a new, processed, 28-kDa virion polypeptide is encoded on the strand complementary to the proposed E1A region. The coding sequences for two other structural proteins are unidentified. The OAV penton protein lacks the region containing an Arg/Gly/Asp sequence that, in human adenoviruses, is thought to interact with cellular integrins to facilitate virus entry. Analysis of proteins and peptides in purified OAV identified several cleavage sites utilized by the Ad proteinase. Some of these were previously identified in human Ad proteins, but new sites, some of which did not conform to the known specificity of the human Ad proteinase, were also identified. The data emphasize that this ovine virus differs significantly from other known human and animal adenoviruses.

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The Escherichia coli enzyme ( purine nucleoside phosphorylase, PNP ) gene is delivered directly into PC3 tumors by one injection of replication -deficient human type -5 adenovirus ( Ad5 ). Expressed PNP converts the systemically administered prodrug, 6MPDR, to a toxic purine, 6MP, causing cell death. We sought to increase the specificity of recombinant Ad vectors by controlling PNP expression with the promoter region from the androgen -dependent, prostate -specific rat probasin ( Pb ) gene. To increase its activity, the promoter was combined with the SV40 enhancer ( SVPb ). Cell lines were transfected with plasmids containing both a reporter gene, under SVPb control, and a reference gene cassette to allow normalization of expression levels. Plasmids expressed $20 -fold more reporter in prostate cancer than in other cells, but surprisingly, the SVPb element was both androgen -independent and retained substantial prostate specificity. Killing by Ad5 -SVPb -PNP vector of cell lines cultured with 6MPDR for 6 days was 5 -to 10 -fold greater in prostate cancer than in liver or lung cells. In vivo, a single intratumoral injection of Ad5 -SVPb -PNP ( 4Â10 8 pfu ), followed by 6MPDR administration twice daily for 6 days, significantly suppressed the growth of human prostate tumors in nude mice and increased their survival compared to control animals. Thus, the androgen -independent, prostate -targeting Ad5 vector reduces human prostate cancer growth significantly in vitro and in vivo. This first example of an androgen -independent vector points the way toward treatment of emerging androgen -independent prostate cancer in conjunction with hormone ablation therapy at a time when the tumor burden is low.

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Up to 25% of colorectal cancer (CRC) may be caused by inherited genetic variants that have yet to be identified. Previous genome-wide linkage studies (GWLSs) have identified a new loci postulated to contain novel CRC risk genes amongst affected families carrying no identifiable mutations in any of the known susceptibility genes for familial CRC syndromes. To undertake a new GWLS, we recruited members from 54 non-syndromic families from Australia and Spain where at least two first-degree relatives were affected by CRC. We used single-nucleotide polymorphism arrays to genotype 98 concordant affected relative pairs that were informative for linkage analyses. We tested for genome-wide significance (GWS) for linkage to CRC using a quantile statistic method, and we found that GWS was achieved at the 5% level. Independently, using the PSEUDO gene-dropping algorithm, we also found that GWS for linkage to CRC was achieved (P¼0.02). Merlin non-parametric linkage analysis revealed significant linkage to CRC for chromosomal region 10p15.3-p15.1 and suggestive linkage to CRC for regions on 14q and 9q. The 10p15.3-p15.1 has not been reported to be linked to hereditary CRC in previous linkage studies, but this region does harbour the Kruppel-like factor 6 (KLF6) gene that is known to be altered in common CRC. Further studies aimed at localising the responsible genes, and characterising their function will give insight into the factors responsible for susceptibility in such families, and perhaps shed further light on the mechanisms of CRC development.