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Published on: June 16, 2011
Isolation and characterization of mycoplasma virus L3 temperature-sensitive mutants
K Haberer1, A I Haberer, S P Cadden
1Department of Microbiology and Immunology, University of Rochester Medical Center, New York 14642.
Abstract:
Mycoplasma virus L3 virions are morphologically similar to coliphage T7, contain linear double-stranded DNA of about 39 kilobase pairs, and produce a nonlytic cytocidal infection in Acholeplasma laidlawii host cells. Following nitrous acid mutagenesis, ninety-eight L3 temperature-sensitive (ts) mutants were isolated from a total of 57,000 plaque-forming units (PFU), using 37 degrees C as the permissive temperature and 41 degrees C as the nonpermissive temperature, with reversion frequencies of 10(-5) to 10(-8). Complementation tests allowed fifty-seven of the L3 ts mutants to be placed into twenty-one complementation groups. In mixed infections, recombination frequencies between mutants in different complementation groups were 10(-2) to less than 10(-6). Studies of protein synthesis in L3-infected cells showed synthesis of about twenty virus-specific proteins, including ten L3 virion proteins. After infection with L3 ts mutants from each complementation group, several different patterns of cell- and virus-specific protein synthesis were observed.
Insights
Researchers isolated 98 temperature-sensitive mutants of Mycoplasma virus L3. Complementation tests grouped these mutants, revealing insights into viral gene function and protein synthesis during nonlytic infections of Acholeplasma laidlawii.
Area of Science:
- Virology
- Molecular Biology
- Microbiology
Background:
- Mycoplasma virus L3 (L3) exhibits morphological similarities to coliphage T7.
- L3 contains linear double-stranded DNA (approx. 39 kbp) and causes nonlytic, cytocidal infections in Acholeplasma laidlawii.
- Understanding L3's genetic makeup and replication is crucial for mycoplasma research.
Purpose of the Study:
- To genetically map Mycoplasma virus L3 using temperature-sensitive (ts) mutants.
- To investigate the roles of L3 genes in viral replication and protein synthesis.
- To establish complementation groups for L3 mutants.
Main Methods:
- Isolation of 98 L3 temperature-sensitive (ts) mutants via nitrous acid mutagenesis.
- Complementation analysis to group ts mutants into functional units.
- Recombination frequency analysis in mixed infections.
- Analysis of viral and host protein synthesis post-infection using L3 ts mutants.
Main Results:
- Fifty-seven L3 ts mutants were assigned to 21 complementation groups.
- Recombination frequencies varied significantly between mutants in different complementation groups.
- Infection with L3 ts mutants resulted in distinct patterns of viral and cell-specific protein synthesis.
- Approximately 20 virus-specific proteins, including 10 virion proteins, were identified.
Conclusions:
- The study successfully generated and characterized a collection of Mycoplasma virus L3 ts mutants.
- Complementation and recombination data provide a framework for understanding L3 gene functions.
- Differential protein synthesis patterns highlight the complex interplay between L3 and its host during infection.
