The broad aim of this proposal is to elucidate the molecular details of an unusual type of non-Mendelian inheritance in Paramecium tetraurelia. We will combine molecular biology and genetics to study a mutant cell line (d48) that contains a complete copy of the A type variable surface antigen gene in the micronucleus but not in the macronucleus. Wild type cytoplasm allows the incorporation of these sequences into the macronucleus, but in d48 cytoplasm the chromosome is broken incorrectly and the A gene is deleted. Recent experiments have shown that macronuclear transformation of d48 with a recombinant clone containing the A antigen gene (pSA14SB) leads to the correct processing of the micronuclear A gene sequences during the formation of the next macronucleus (K. Aufderheide, personal communication). By microinjecting specific fragments of pSA14SB into the d48 macronucleus we will define the DNA sequences necessary for the correct processing of the A gene. Further experiments will be performed to determine if RNA products from these sequences are important for their function. Micronuclear DNA from the d48 and wild type cell lines will be isolated and used to compare the organization of the micronuclear sequences surrounding the chromosome breakage sites. Additional non-Mendelian mutants have recently been isolated that affect expression of the B surface antigen gene (J. Preer, personal communication). We will clone the B gene and determine if similar molecular mechanisms are involved in these mutations. We believe this research will advance the understanding of developmentally controlled DNA rearrangements in eukaryotes, and perhaps reveal novel aspects of nucleic acid processing.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29GM043357-03
Application #
3467963
Study Section
Genetics Study Section (GEN)
Project Start
1989-12-01
Project End
1994-11-30
Budget Start
1991-12-01
Budget End
1992-11-30
Support Year
3
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Purdue University
Department
Type
Schools of Earth Sciences/Natur
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
Leeck, C L; Forney, J D (1994) The upstream region is required but not sufficient to control mutually exclusive expression of Paramecium surface antigen genes. J Biol Chem 269:31283-8
Scott, J; Leeck, C; Forney, J (1994) Analysis of the micronuclear B type surface protein gene in Paramecium tetraurelia. Nucleic Acids Res 22:5079-84
Scott, J M; Mikami, K; Leeck, C L et al. (1994) Non-Mendelian inheritance of macronuclear mutations is gene specific in Paramecium tetraurelia. Mol Cell Biol 14:2479-84
Asai, D J; Beckwith, S M; Kandl, K A et al. (1994) The dynein genes of Paramecium tetraurelia. Sequences adjacent to the catalytic P-loop identify cytoplasmic and axonemal heavy chain isoforms. J Cell Sci 107 ( Pt 4):839-47
You, Y; Scott, J; Forney, J (1994) The role of macronuclear DNA sequences in the permanent rescue of a non-mendelian mutation in Paramecium tetraurelia. Genetics 136:1319-24
Scott, J; Leeck, C; Forney, J (1993) Molecular and genetic analyses of the B type surface protein gene from Paramecium tetraurelia. Genetics 134:189-98
Forney, J; Rodkey, K (1992) A repetitive DNA sequence in Paramecium macronuclei is related to the beta subunit of G proteins. Nucleic Acids Res 20:5397-402
Nielsen, E; You, Y; Forney, J (1991) Cysteine residue periodicity is a conserved structural feature of variable surface proteins from Paramecium tetraurelia. J Mol Biol 222:835-41
You, Y; Aufderheide, K; Morand, J et al. (1991) Macronuclear transformation with specific DNA fragments controls the content of the new macronuclear genome in Paramecium tetraurelia. Mol Cell Biol 11:1133-7