Alteration of Open Reading Frames by Use of New Gene Cassettes

ABSTRACT The vast majority of bacteria in the environment have yet to be cultured. Consequently, a major proportion of both genetic diversity within known gene families and an unknown number of novel gene families reside in these uncultured organisms. Isolation of these genes is limited by lack of sequence information. Where such sequence data exist, PCR directed at conserved sequence motifs recovers only partial genes. Here we outline a strategy for recovering complete open reading frames from environmental DNA samples. PCR assays were designed to target the 59-base element family of recombination sites that flank gene cassettes associated with integrons. Using such assays, diverse gene cassettes could be amplified from the vast majority of environmental DNA samples tested. These gene cassettes contained complete open reading frames, the majority of which were associated with ribosome binding sites. Novel genes with clear homologies to phosphotransferase, DNA glycosylase, methyl transferase, and thiotransferase genes were identified. However, the majority of amplified gene cassettes contained open reading frames with no identifiable homologues in databases. Accumulation analysis of the gene cassettes amplified from soil samples showed no signs of saturation, and soil samples taken at 1-m intervals along transects demonstrated different amplification profiles. Taken together, the genetic novelty, steep accumulation curves, and spatial heterogeneity of genes recovered show that this method taps into a vast pool of unexploited genetic diversity. The success of this approach indicates that mobile gene cassettes and, by inference, integrons are widespread in natural environments and are likely to contribute significantly to bacterial diversity.

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Promoter activity of ORF-less gene cassettes isolated from the oral metagenome

10.1101/427781 ◽

2018 ◽

Author(s):

Supathep Tansirichaiya ◽

Peter Mullany ◽

Adam P. Roberts

Keyword(s):

Promoter Activity ◽

Detection System ◽

Agar Plate ◽

Open Reading Frames ◽

Direct Identification ◽

Gene Cassettes ◽

Promoter Sequences ◽

Antisense Mrna ◽

Physico Chemical ◽

Reading Frames

AbstractIntegrons are genetic elements consisting of a functional platform for recombination and expression of gene cassettes (GCs). GCs usually carry promoter-less open reading frames (ORFs), encoding proteins with various functions including antibiotic resistance. The transcription of GCs relies mainly on a cassette promoter (PC), located upstream of an array of GCs. Some integron GCs, called ORF-less GCs, contain no identifiable ORF with a small number shown to be involved in antisense mRNA mediated gene regulation.In this study, promoter sequences were identified, usingin silicoanalysis, within GCs PCR amplified from the oral metagenome. The promoter activity of ORF-less GCs was verified by cloning them upstream of agusAreporter, proving they can function as a promoter, presumably allowing bacteria to adapt to multiple stresses within the complex physico-chemical environment of the human oral cavity. A bi-directional promoter detection system was also developed allowing direct identification of clones with promoter-containing GCs on agar plates. Novel promoter-containing GCs were identified from the human oral metagenomic DNA using this construct, called pBiDiPD.This is the first demonstration and detection of promoter activity of ORF-less GCs and the development of an agar plate-based detection system will enable similar studies in other environments.

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Complete Genomic Sequence and Comparative Analysis ofthe Tumorigenic Poxvirus Yaba Monkey TumorVirus

Journal of Virology ◽

10.1128/jvi.77.24.13335-13347.2003 ◽

2003 ◽

Vol 77 (24) ◽

pp. 13335-13347 ◽

Cited By ~ 37

Author(s):

Craig R. Brunetti ◽

Hiroko Amano ◽

Yoshiaki Ueda ◽

Jing Qin ◽

Tatsuo Miyamura ◽

...

Keyword(s):

Comparative Analysis ◽

Genomic Sequence ◽

Tumor Formation ◽

Open Reading Frames ◽

Noncoding Regions ◽

Conserved Sequence ◽

Tanapox Virus ◽

Tumor Virus ◽

New Gene ◽

Reading Frames

ABSTRACT The Yatapoxvirus genus of poxviruses is comprised of Yaba monkey tumor virus (YMTV), Tanapox virus, and Yaba-like disease virus (YLDV), which all have the ability to infect primates, including humans. Unlike other poxviruses, YMTV induces formation of focalized histiocytomas upon infection. To gain a greater understanding of the Yatapoxvirus genus and the unique tumor formation properties of YMTV, we sequenced the 134,721-bp genome of YMTV. The genome of YMTV encodes at least 140 open reading frames, all of which are also found as orthologs in the closely related YLDV. However, 13 open reading frames found in YLDV are completely absent from YMTV. Common to both YLDV and YMTV are the unusually large noncoding regions between many open reading frames. To determine whether any of these noncoding regions might be functionally significant, we carried out a comparative analysis between the putative noncoding regions of YMTV and similar noncoding regions from other poxviruses. This approach identified three new gene poxvirus families, defined as orthologs of YMTV23.5L, YMTV28.5L, and YMTV120.5L, which are highly conserved in virtually all poxvirus species. Furthermore, the comparative analysis also revealed a 40-bp nucleotide sequence at approximately 14,700 bases from the left terminus that was 100% identical in the comparable intergene site within members of the Yatapoxvirus, Suipoxvirus, and Capripoxvirus genera and 95% conserved in the Leporipoxvirus genus. This conserved sequence was shown to function as a poxvirus late promoter element in transfected and infected cells, but other functions, such as an involvement in viral replication or packaging, cannot be excluded. Finally, we summarize the predicted immunomodulatory protein repertoire in the Yatapoxvirus genus as a whole.

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