Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-05-23
1998-10-13
Railey, II, Johnny F.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 691, 4351723, 4352523, 4353201, 536 241, C12Q 102, C12N 121, C12N 1510, C12N 1511
Patent
active
058210598
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
This application is the U.S. national stage application of PCT/US93/07407 filed under 35 U.S.C. 371.
The class Mollicutes encompasses a group of organisms collectively known as "mycoplasmas," many of which are important human and agricultural pathogens. Despite this pathogenicity, little is known about the genetics of mycoplasmas. These organisms possess the smallest genome thought necessary for autonomous existence. Razin, Microbiol. Rev. 49: 419-55 (1985). Due to their simplicity, most mycoplasma species require complex media for growth because they lack many biosynthetic pathways. In view of such limitations, traditional genetic studies employing auxotrophic mutants have not been possible with these organisms.
Based on RNA homology, mycoplasmas are thought to be a product of degenerative evolution from Gram-positive organisms. Previous studies of 16S rRNA sequence homology have suggested that mycoplasmas are more closely related to Gram-positive organisms than Gram-negative organisms Weisburg et al., J. Bacteriol. 171: 6455-67 (1989). The differences in translational specificity that have been demonstrated between the Gram-negative and Gram-positive bacteria also appear to pertain to mycoplasmas as well. Hager & Rabinowitz, The Molecular Biology of the Bacilli 1-34 (Dubnau ed., Acad. Press 1985).
The simplicity of mycoplasmas offers advantages in the context of expression systems. For example, mycoplasmas lack lipopolysaccharide and other toxic wall constituents, which would allow for simplified purification of recombinantly produced proteins. Significant problems have existed, however, with using mycoplasmas as a recombinant expression system. Adequate stability of cloned genes has previously not been achieved. Moreover, previous attempts at creating mycoplasma-based expression systems have employed gram-negative promoters, which was necessitated by the unavailability and limited knowledge regarding mycoplasma promoters, generally. The transcriptional apparatus of gram-negative bacteria, however, is often unable to correctly recognize mycoplasma promoter sequences. For instance, it has been shown that, although the rRNA promoter of Mycoplasma capricolum is recognized by both E. coli and M. capricolum RNA polymerase, it is not properly recognized in E. coli. Gafny et al., Nucl. Acids Res. 16: 61-76 (1988). Thus, the recognition of the mycoplasma rRNA promoter is activated in E. coli under the stringent condition of amino acid starvation, which is opposite of the expected result. Additional problems exist with such use of gram-negative hosts. Signals may arise from transcription initiation at pseudo-promoter sites, which are caused by the high (A+T) content in the mycoplasma DNA of the fusion gene. Vollenweider et al., Science 205: 508-11 (1979). Notarnicola et al. have shown that E. coli initiated translation at internal sites in a Mycoplasma hyorhinis lipoprotein structural gene. F. Biol. 172: 2986-95 (1990). It has become apparent, therefore, that the use of E. coli as a cloning host to study promoter sequences from organisms with a high (A+T) content, such as mycoplasmas, should be limited.
The lack of genetic tools also has made the development of mycoplasma cloning systems difficult. Only two transposons, Tn916 and Tn4001, have been shown to be useful for studying mycoplasma genetics. Dybvig & Alderete, Plasmid 20: 33-41 (1988); Dybvig & Cassell, Science 235: 1392-94 (1987); Mahairas & Minion, Plasmid 21: 43-47 (1989). A number of broad host-range plasmids from Gram-positive bacteria have been examined as possible cloning vectors, but all have proven to be unstable. Dybvig, Plasmid 21: 155-60 (1989). Naturally occurring mycoplasma plasmids have also been examined as possible cloning vectors, but they have not been shown to maintain and express a cloned gene. Dybvig et al., IOM Letts. 1: 209-10 (1990); King & Dybvig, Plasmid 28: 86-91 (1992). A cloning system has been developed in spiroplasmas which uses a spiroplasma virus as a cloning vector, but the vector has a limit
REFERENCES:
Loechel et al., Nucleic Acids Research 19 (24): 6905-6912 (1991).
Mahairas et al., "Random Insertion of the Gentamicin Resistance Transposon Tn4001 In Mycoplasma Pulmonis", Plasmid, vol. 21:43-47, (1989).
Mahairas et al., "Development of A Cloning System In Mycoplasma Plumonis", Gene, vol. 93:61-65, (1990).
Dybvig, "Transformation of Acholeplasma Laidlawii With Streptococcal Plasmids pVA868 and pVA920", Plasmid, vol. 21:155-160, (1989).
Gafney et al., "Promoters of Mycoplasma Capricolum Ribosomal RNA Operons: Identical Activities But Different Regulation In Homologous And Heterologous Cells", Nucl. Acids, Res., vol. 16:61-76, (1988).
Rodriguez et al., "A Survey of Molecular Cloning Vectors and Their Uses", Vectors, Butterworths, pp. 153-177, (1988).
Knudtson Kevin L.
Minion F. Chris
Iowa State University & Research Foundation, Inc.
Railey II Johnny F.
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